Receptor tyrosine kinase

ABSTRACT

Novel receptor tyrosine kinase protein and isoforms thereof which are expressed in cells of the endothelial lineage, and DNA segments encoding the novel protein and isoforms thereof are disclosed. Methods for identifying ligands which are capable of binding to the receptor protein and methods for screening for agonist or antagonist substances of the interaction of the protein and a ligand are also disclosed.

This application is a division of application Ser. No. 08/278,089, filedJul. 20, 1994, now U.S. Pat. No. 5,681,714, which is acontinuation-in-part of U.S. Ser. No. 08/235,408, filed Apr. 29, 1994,now abandoned, which is a continuation-in-part of U.S. Ser. No.07/921,795, filed Jul. 30, 1992, now abandoned.

FIELD OF THE INVENTION

The invention relates to a novel receptor tyrosine kinase protein,isoforms and parts thereof, nucleic acid molecules encoding the novelprotein and fragments thereof, and uses of the protein and nucleic acidmolecules.

BACKGROUND OF THE INVENTION

Transmembrane receptor tyrosine kinases (RTKs) comprise a large andevolutionarily conserved family of structurally related proteins capableof transducing extracellular signals to the cytoplasm. The latentoncogenic potential of these molecules and the molecular mechanisms bywhich they function in signalling pathways have been the subject ofextensive study.

In addition, genetic and biochemical analyses of a variety ofdevelopmental mutants have led to recognition of the pivotal rolesplayed by RTK-mediated signalling pathways in the regulation of celldetermination, migration, and proliferation. Notable examples inDrosophila include the role of sevenless and its ligand, bride ofsevenless, in R7 photoreceptor determination (Kramer, H., Cagan, R. L. &Zipursky, S. L. (1991), Nature, 352, 207-212), and of DER/flb in earlymorphogenetic events during gastrulation (Schejter, E. D. & Shilo, B.-Z.(1989), Cell, 56, 1093-1104). Similarly, in the mouse, loss of functionmutations at the W/c-kit (Geissler, E. N., Rayn, M. A. & Housman, D. E.(1988), Cell, 55, 185-192; Chabot, B., Stephenson, D. A., Chapman, V.M., Besmer, P. & Bernstein, A. (1988), Nature, 335, 88-89) and Sl(Russell, E. S. (1979), Adv.Genet., 20, 357-459) loci have revealed theimportance of the Kit receptor and its ligand in melanogenesis,hematopoiesis, and gametogenesis (Dubreuil, P., Rottapel, R., Reith, A.D., Forrester, L. & Bernstein, A. (1990), Ann. N.Y. Acad. Sci., 599,58-65; Williams, D. E., Eisenman, J., Baird, A., Rauch, C., Ness, K. V.,March, C. J., Park, L. S., Martin, U., Mochizuki, D. Y., Boswell, H. S.,Burgess, G. S., Cosman, D. & Lyman, S. D. (1990), Cell, 63, 167-174;Copeland, N. G., Gilbert, D. J., Cho, B. C., Donovan, P. J., Jenkins, N.A., Cosman, D. Anderson, D., Lyman, S. D. & Williams, D. E. (1990),Cell, 63, 175-183 and Flanagan, J. G. & Leder, P. (1990), Cell, 63,185-194) while a deletion in the gene encoding PDGFR-α has beencorrelated with the Patch mutation, which also causes a defect inmelanogenesis (Stephenson, D. A., Mercola, M., Anderson, E., Wang, C.,Stiles, C. D., Bowen-Pope, D. F. & Chapman, V. M. (1991),Proc.Natl.Acad.Sci., 88, 6-10). These observations, together with others(reviewed in Pawson, T. & Bernstein, A. (1991), Trends Gen., 6,350-356), have established the importance of receptor-ligandinteractions in the regulation of development.

Angiogenesis in both the embryo and adult requires the differentiation,proliferation, and migration of endothelial cells. Tissuetransplantation studies with quail/chick chimeras have established thatthe developmental cues for both endothelial cell differentiation andproper patterning of vessels are extracellular and not pre-programmedwithin the cell (Noden, D. M. (1988) Development, 103, 121-140) Severalpeptide hormones, such as bFGF, VEGF and PD-EGF, have been shown to haveboth mitogenic and chemotactic effects on cultured endothelial cells(see Tomasi, V., Manica, F. & Spisni, E. (1990), BioFactors, 2, 213-217;Klagsbrun, M. & D'Amore, P. (1991), Annu.Rev.Physiol., 53, 217-239, forreviews). However, many of these factors also show similar effects onother cell types, implying that receptors for these factors are alsoexpressed by such cells.

Studies have demonstrated that both tyrosine kinase activity andphosphotyrosine-containing proteins are increased in embryonic chickenheart relative to the adult (Maher, P. A. (1991). J.Cell Biol., 112,955-963), and that inhibitors of kinase activity impede inductiveprocesses during in vitro differentiation of cardiac explants derivedfrom chicken embryos (Runyan, R. B., Potts, J. D., Sharma, R. V.,Loeber, C. P., Chiang, J. J. & Bhalla, R. C. (1990), Cell Reg., 1,301-313).

SUMMARY OF THE INVENTION

The present inventors have identified and characterized a receptortyrosine kinase protein that plays a critical role in murinecardiogenesis. The heart forms early in mouse embryogenesis and itsdevelopment is known to be accompanied by the differentiation frommesoderm of myocytes and endothelial cells that subsequently form themyocardium and endocardium, respectively (Manasek, F. J. (1976), in TheCell Surface in Animal Embryogenesis and Development, p.545-598,Elsevier/North-Holland Biomedical Press; Kaufman, M. H. & Navaratnam, V.(1981), J.Anat., 133, 235-246). There have not hitherto been any reportsof directed screens for tyrosine kinases expressed during murinecardiogenesis.

In particular, the present inventors using reverse transcription coupledto the polymerase chain reaction (RT-PCR) isolated from murine embryonicheart a cDNA, designated tek, whose deduced amino acid sequencecorresponds to a novel RTK. The tek locus of mouse was mapped tochromosome 4. The present inventors have also shown by in situhybridization that tek is expressed in the endocardium as well as theendothelial lining of the vasculature. tek was also found to beexpressed in both mature endothelial cells and their progenitors,suggesting that the signalling pathways regulated by tek may beimportant to both the determination and proliferation of cells of theendothelial lineage. The tek locus of humans was mapped to the humanchromosome 9p21 region. This region is deleted or rearranged in manytypes of neoplasia, suggesting that the tek locus may play a role inoncogenesis.

The present inventors have cloned and sequenced a 4.2-kb murine cDNAencoding the novel receptor tyrosine kinase. Conceptual translation ofthe 4.2-kb cDNA revealed a single large open reading frame from aputative initiation codon at nucleotide 124 to an in-frame stop codon atnucleotide 3490. The inventors have determined the primary structure ofthe deduced receptor tyrosine kinase protein. The 1,122 residuepolypeptide corresponds to a receptor tyrosine kinase protein containinga kinase region interrupted by a 21 amino acid insert linked via atransmembrane domain to a remarkably complex novel extracellular domain.The extracellular domain comprises three Fibronectin type III (FNIII)repeats, immediately following the transmembrane domain, fused to twoimmunoglobulin-like (Ig-like) loops that are themselves separated bythree tandem epidermal growth factor-like (EGF-like) repeats.

The present inventors have also demonstrated that the 4.2-kb cDNAencodes a 140-kDa protein that co-migrates with a polypeptidespecifically detected by antibody directed against the novel receptortyrosine kinase protein in both cultured endothelial cells and highlyvascularized embryonic tissues. A 140-kDa protein was also specificallyprecipitated from cells transfected with the cDNA.

The present inventors have further elucidated the role of the novelreceptor tyrosine kinase within the endothelial cell lineage bydisrupting its signalling pathway using two different geneticapproaches. First, transgenic mice expressing a dominant-negative formof the novel receptor tyrosine kinase protein were constructed. Second,a null allele of the tek locus was created by homologous recombinationin embryonic stem cells. Transgenic mice expressing dominant-negativealleles of tek or homozygous for the null allele of the tek locus bothdied in utero. Analysis of mice carrying either dominant-negative ornull mutations of the tek gene confirmed that the tek signalling pathwayplays a critical role in the differentiation, proliferation and survivalof endothelial cells in the mouse embryo.

The present invention therefore provides a purified and isolated nucleicacid molecule, preferably a DNA molecule, having a sequence which codesfor a receptor tyrosine kinase protein which is expressed in cells ofendothelial lineage, or an oligonucleotide fragment of the nucleic acidmolecules which is unique to the receptor tyrosine kinase protein of theinvention. In a preferred embodiment of the invention, the purified andisolated nucleic acid molecule has the sequence as shown in SEQ ID NO:1and in SEQ ID NO:5.

The invention also contemplates a double stranded nucleic acid moleculecomprising a nucleic acid molecule of the invention or anoligonucleotide fragment thereof hydrogen bonded to a complementarynucleotide base sequence.

The present invention provides in one embodiment, an isolated andpurified nucleic acid molecule comprising: (a) a sequence encoding aprotein having the amino acid sequence as shown in SEQ ID NO:6 and FIG.11B, wherein T can also be U; (b) nucleic acid sequences complementaryto (a); (c) nucleic acid sequences which are at least 95% homologous to(a); or, (d) a fragment of (a) or (b) that is at least 18 bases andwhich will hybridize to (a) or (b) under stringent conditions. In aparticular embodiment, the fragment is a sequence encoding a receptortyrosine kinase extracellular domain having the amino acid sequence asshown in SEQ ID NO:6 from amino acid number 19 to 744 and sequenceshaving at least 97% homology thereto.

The present invention also provides a purified and isolated nucleic acidmolecule comprising: (a) a sequence as shown in SEQ ID NO: 5 and FIG.11B; (b) nucleic acid sequences complementary to (a); (c) nucleic acidsequences which are at least 95% homologous to (a); or, (d) a fragmentof (a) or (b) that is at least 18 bases and which will hybridize to (a)or (b) under stringent conditions.

It is contemplated that a nucleic acid molecule of the invention may beprepared having a structural mutation including, replacement, deletionor insertion mutations. For example, the signal peptide may be deleted,in particular, the first 17 amino acids of tek as shown in SEQ ID NO. 6and FIG. 11B, may be deleted. As another example, lysine⁸⁵³ toalanine⁸⁵³ may be altered to generate a protein that is still competentto bind ligand, but which is catalytically inactive and thus unable totransduce a signal.

The invention further contemplates a recombinant molecule comprising anucleic acid molecule of the invention or an oligonucleotide fragmentthereof and an expression control sequence operatively linked to thenucleic acid molecule or oligonucleotide fragment. A transformant hostcell including a recombinant molecule of the invention is also provided.

Still further, this invention provides plasmids which comprise thenucleic acid molecules of the invention.

The invention further provides a method of preparing a novel receptortyrosine kinase protein or isoforms thereof utilizing the purified andisolated nucleic acid molecule of the invention. The method comprisesculturing a transformant host cell including a recombinant moleculecomprising a nucleic acid molecule of the invention and an expressioncontrol sequence operatively linked to the nucleic acid molecule, in asuitable medium until the protein is formed and thereafter isolating theprotein.

The invention further broadly contemplates a substantially pure receptortyrosine kinase protein or a part thereof, which is expressed in cellsof endothelial lineage.

The receptor tyrosine kinase protein of the invention is furthercharacterized as containing an extracellular domain comprising at leastone fibronectin III repeat, at least one immunoglobulin-like loop and atleast one epidermal growth factor-like repeat. The extracellular domaincomprises three fibronectin III repeats, two immunoglobulin-like loopsand three fibronectin III repeats. The three fibronectin III repeats arefused to the two immunoglobulin-like loops and the twoimmunoglobulin-like loops are separated by the three fibronectin IIIrepeats.

In an embodiment, the invention provides a purified and isolated proteinhaving an amino acid sequence as shown in SEQ ID NO: 6 or a sequencehaving at least 97% homology thereto, or a part of the protein having atleast 20 amino acids. The part of the protein preferably comprises anextracellular domain of a receptor tyrosine kinase having the amino acidsequence as shown in SEQ ID NO: 6 from amino acid number 19 to 744 or asequence having at least 97% homology thereto. Conjugates of the Tekprotein of the invention, or parts thereof may be prepared. This may beaccomplished, for example by the synthesis of N-terminal or C-terminalfusion proteins. The invention therefore also relates to fusion proteinscomprising a part of the protein as described herein and, optionally amarker protein, such as the Fc portion of an immunoglobulin.

The present invention also includes a receptor tyrosine kinase proteinof the invention or part thereof, preferably the catalytic domain, whichis enzymatically active. The catalytically active form of the protein orpart thereof is also referred to herein as an "activated receptortyrosine kinase protein or part thereof".

The invention further contemplates antibodies having specificity againstan epitope of the receptor tyrosine kinase protein of the invention orpart of the protein. Antibodies may be labelled with a detectablesubstance and they may be used to detect the novel receptor tyrosinekinase of the invention in tissues and cells. The antibodies maytherefore be used to monitor angiogenesis, cardiogenesis andtumorigenesis.

The invention also permits the construction of nucleotide probes whichare unique to the novel receptor tyrosine kinase protein of theinvention or a part of the protein. Thus, the invention also relates toa probe comprising a nucleotide sequence coding for a protein, whichdisplays the properties of the novel receptor tyrosine kinase of theinvention or a peptide unique to the protein. The probe may be labelled,for example, with a radioactive substance and it may be used to selectfrom a mixture of nucleotide sequences a nucleotide sequence coding fora protein which displays the properties of the novel receptor tyrosinekinase protein of the invention.

The present invention also provides a transgenic non-human animal orembryo all of whose germ cells and somatic cells contain a recombinantmolecule of the invention preferably a recombinant molecule comprisingthe nucleic acid molecules of the invention containing a sequenceencoding the receptor tyrosine kinase protein of the invention or partthereof with a structural mutation or comprising the nucleic acidmolecules of the invention containing a sequence encoding the receptortyrosine kinase protein of the invention or part thereof and one or moreregulatory elements which differ from the regulatory elements of thenative protein.

The invention still further provides a method for identifying asubstance, which is capable of binding to the novel receptor tyrosinekinase protein of the invention, comprising reacting the novel receptortyrosine kinase protein of the invention or part of the protein underconditions which permit the formation of a complex between the substanceand the novel receptor tyrosine kinase protein or part of the proteinand assaying for substance-receptor complexes, for free substance, fornon-complexed receptor tyrosine kinase protein, or for activation of thereceptor tyrosine kinase protein.

An embodiment of the invention provides a method for identifying ligandswhich are capable of binding to the novel receptor tyrosine kinaseprotein of the invention, isoforms thereof, or part of the protein,comprising reacting the novel receptor kinase protein of the invention,isoforms thereof, or part of the protein, with at least one ligand whichpotentially is capable of binding to the protein, isoform or part of theprotein, under conditions which permit the formation of ligand-receptorprotein complexes, and assaying for ligand-receptor protein complexes,for free ligand, for non-complexed proteins or for activation of thereceptor tyrosine kinase protein. In a preferred embodiment of themethod, ligands are identified which are capable of binding to andactivating the novel receptor tyrosine kinase protein of the invention,isoforms thereof, or part of the protein. The ligands which bind to andactivate the novel receptor tyrosine kinase receptor of the inventionare identified by assaying for protein tyrosine kinase activity i.e. byassaying for phosphotyrosine.

In addition, the invention provides a method of using the novel proteinsof the invention for assaying a medium for the presence of a substancethat affects a tek effector system. In accordance with one embodiment, amethod is provided which comprises providing a known concentration of areceptor tyrosine kinase protein of the invention, or a part thereof,incubating the protein, or a part thereof, with a substance which iscapable of binding to the protein or part thereof, and therebyactivating the tek effector system, and a suspected agonist orantagonist substance under conditions which permit the formation ofligand-receptor protein complexes, and assaying for ligand-receptorprotein complexes, for free ligand or for non-complexed protein or foractivation of the receptor tyrosine kinase protein.

The invention also relates to a method for assaying a medium for thepresence of an agonist or antagonist of the interaction of the novelreceptor tyrosine kinase protein and a substance which is capable ofbinding to the receptor tyrosine kinase protein, which comprisesproviding a known concentration of the receptor tyrosine kinase protein,reacting the receptor tyrosine kinase protein with a substance which iscapable of binding to the receptor tyrosine kinase protein and asuspected agonist or antagonist under conditions which permit theformation of substance-receptor tyrosine kinase complexes, and assayingfor substance-receptor tyrosine kinase complexes, for free substance,for non-complexed proteins, or for activation of the receptor tyrosinekinase.

The methods of the invention make it possible to screen a large numberof potential ligands for their ability to bind to the novel receptortyrosine kinase protein of the present invention. The methods of theinvention will also be useful for identifying substances which mayaffect cardiogenesis and angiogenesis and/or maintenance of cells of theendothelial lineage and which may play a role in tumorigenesis.

Substances which affect angiogenesis, cardiogenesis or tumorigenesis maybe identified using the methods of the invention by comparing thepattern and level of expression of the novel receptor tyrosine kinaseprotein of the invention in tissues and cells in the presence and in theabsence of the substance.

The invention further contemplates a method for identifying a substancewhich is capable of binding to an activated receptor tyrosine kinaseprotein of the invention or an isoform or part of the activated protein,comprising reacting an activated receptor tyrosine kinase protein of theinvention, or an isoform, or part of the protein, with at least onesubstance which potentially can bind with the receptor tyrosine kinaseprotein, isoform or part of the protein, under conditions which permitthe formation of substance-receptor kinase protein complexes, andassaying for substance-receptor kinase protein complexes, for freesubstance, for non-complexed receptor kinase proteins, or forphosphorylation of the substance. The method may be used to identifyintracellular ligands such as Src homology region 2 (SH2) containingproteins which bind to an activated receptor tyrosine kinase of theinvention or parts thereof or intracellular ligands which may bephosphorylated by the protein.

DESCRIPTION OF THE DRAWINGS

The invention will be better understood with reference to the drawingsin which:

FIG. 1 shows a nucleotide and deduced amino acid sequence of a receptortyrosine kinase protein of the invention as shown in SEQ ID NOS:1 and 2;

FIG. 2 shows a nucleotide and deduced amino sequence of a 1601 bp DNAmolecule of the invention as shown in SEQ ID NOS:3 and 4;

FIG. 3 shows a comparison of a portion of the deduced amino acidsequence of the novel receptor tyrosine kinase protein of the invention(SEQ ID NO:4) with that of other tyrosine kinases (SEQ ID NO:1, 14 to16);

FIG. 4 shows a Northern blot hybridization analysis of expression of aDNA molecule of the invention in 12.5 day murine embryonic heart;

FIG. 5 shows the in situ hybridization analysis of expression of a DNAmolecule of the invention in the 12.5 day embryo, (A) dark fieldillumination of a para-sagittal section, (B) and (C) bright and darkfield illumination respectively, of a mid saggital section through theheart region;

FIG. 6 shows the expression of a DNA molecule of the invention precedesthat of von Willebrand factor in 8.5 day embryos, (A) bright fieldillumination, (B) dark field illumination, (C) blood island, (D) showingabsence of expression of von Willebrand factor in the embryo, (E)showing expression of von Willebrand factor in the endothelial lining ofthe blood vessels of the maternal decidua, (F) cephalic region, (G)saggital section (H) dark field illumination of (G), (I) heart region,(J) tek-expressing cells beneath the ventral surface of the somites;

FIG. 7 shows expression of a DNA molecule of the invention in wholemount embryos(A, B, and C); expression in Day 8.0 embryos (D); mRNAdistribution in a Day 9.5 embryo (E); and En2 expression in a Day 8embryo (F);

FIG. 8 shows the expression of a DNA molecule of the invention precedesthat of von Willebrand factor in the developing leptomeninges and inparticular the absence of immunohistochemical staining of von Willebrandfactor in Day 12.5 leptomeninges (A); in situ detection of tekexpression in Day 12.5 leptomeninges(B); staining of von Willebrandfactor in Day 14.5 leptomeninges (C);

FIG. 9 shows the expression of a nucleic acid molecule of the inventionin adult vasculature and in particular bright field illumination of asection through the upper heart region of a 3 week-old mouse hybridizedwith an [³⁵ S]-labelled probe (A); bright field illumination showingexpression in endothelial cells lining the artery and vein respectively(B) and (C);

FIG. 10 shows the hierarchy of the endothelial cell lineage;

FIG. 11A shows the cDNAs used to assemble the tek cDNA;

FIG. 11B shows the nucleotide and deduced amino acid sequence of a4177-nucleotide tek cDNA as shown in SEQ ID NO: 4 and 5;

FIG. 12A shows a sequence comparison of Tek receptor tyrosine kinaseprotein (SEQ ID NO:17 to 19) and Tie EGF-like repeats (SEQ ID NO:20-22);

FIG. 12B shows a sequence comparison of Tek receptor tyrosine kinaseprotein (SEQ ID NO:25, 27, 29) and Tie fibronectin type III repeats (SEQID NO:26, 28, and 30);

FIG. 13A shows the structural relationship between Tek and Tie by acomparison of structural motifs;

FIG. 13B shows the structural relationship between Tek and Tie bySouthern analysis;

FIG. 14 shows tek and Flk-1 expression in cell lines of endothelialorigin;

FIG. 15A shows that tek directs synthesis of a 140-kDa protein byimmunoprecipitation with anti-tek serum;

FIG. 15B shows that tek directs synthesis of a 140-kDa protein byWestern analysis;

FIG. 16 shows a G-banded partial metaphase spread with silver grain at9p21 (arrow), and;

FIG. 17 shows silver grain distribution on a human karyotype followingin situ hybridization with a tek probe;

FIG. 18A is a schematic showing the transgene used to drive theexpression of the dominant-negative mutant tek^(A853) cDNA;

FIG. 18B is a gel showing that tek^(A853) protein (DN) is catalyticallyinactive compared to wild type (WT) tek protein;

FIG. 19 shows developmentally delayed tek^(A853) transgenic embryos asfollows: 19A shows a non-transgenic control, 19B shows a tek promoterdevelopmentally delayed embryo and 19C shows a polyoma drivendevelopmentally delayed embryo;

FIG. 20A is a schematic showing the strategy used to disrupt the codingsequence of the first exon of the tek gene, generating the mutationtek.sup.Δsp ;

FIG. 20B shows the presence of a tek.sup.Δsp specific fragment (Trg) andwild type tek (wt) in DNA from day 9.5 embryos from a tek.sup.Δsp/+heterozygous F1 intercross;

FIG. 21 shows a histological examination of the heart region of mouseE9.5 transgenic embryos as follows: 21A shows an embryo, containing thetek^(A853) transgene driven by the tek-promoter, 21B shows an embryocontaining the tek^(A853) transgene driven by the polyoma earlysequence, 21C shows tek.sup.Δsp heterozygous embryos and 21D showstek.sup.Δsp homozygous embryos;

FIG. 22 shows a histological analysis of tek.sup.Δsp mutant embryos andnormal littermates as follows: 21A shows the embryonic portion of theplacenta from heterozygous embryos, 21B shows the dorsal aortic regionof heterozygous embryos, 21C shows the yolk sac of heterozygous embryos,21D shows the embryonic portion of the placenta from tek.sup.Δsphomozygous embryos, 21E shows the dorsal aortic region of homozygousembryos and, 21F shows the yolk sac of homozygous embryos.

FIG. 23 shows tek-promoter-lacZ expression in the yolk sac vasculatureof normal and tek.sup.Δsp homozygous embryos as follows: 23A showsexpression in Day 8.5 normal embryos, 23B shows expression in Day 9.0normal embryos, 23C shows expression in Day 8.5 homozygous mutants and,23D shows expression in Day 9.0 homozygous mutants;

FIG. 24 shows tek-promoter-lacz expression in the embryonic vasculatureof normal and tek.sup.Δsp homozygous embryos as follows: 24A showsexpression in the trunk region of E9.0 tek.sup.Δsp homozygous embryos,24B shows expression in the heart region of E9.0 tek.sup.Δsp homozygousembryos, 24C shows expression in the trunk region of wild type embryosand, 24D shows expression in the heart region of wild type embryos and;

FIG. 25 shows expression of the tek-promoter-lacZ transgene intek.sup.Δsp homozygous embryos as follows: 25A shows expression in theendothelial cells of E8.5 wild type embryos, 25B shows expression in theendothelial cells of E9.0 wild type embryos, 25C shows expression in theendothelial cells of E8.5 tek.sup.Δsp homozygous embryos and 25D showsexpression in the endothelial cells of E9.0 tek.sup.Δsp homozygousembryos.

DETAILED DESCRIPTION OF THE INVENTION

I. Characterization of Nucleic Acid Molecules and Proteins of theInvention

The present inventors have isolated a gene encoding a novel receptortyrosine kinase protein, designated tek, expressed during murinecardiogenesis. By analysing the segregation of an AccI restriction sitepolymorphism in AKR/J:DBA recombinant inbred mice, the present inventorsmapped the tek locus to chromosome 4, between the brown and pmv-23 loci.This region is syntenic with human chromosomal regions 1p22-23, 9q31-33,and 9p22-13. In mice and humans, these regions do not contain anypreviously described loci known to be involved with the biology of theendothelial cell lineage (Lyon, M. F. & Searle, A. G. Genetic Variantsand Strains of the Laboratory Mouse, New York:Oxford University Press,1989, 2nd, Ed.; O'Brien, 1990).

The human tek locus was mapped, by the present inventors, to humanchromosome 9p21, a region which is deleted or rearranged in many typesof neoplasia (Fountain et al., 1992; Taguchi et al., 1993; Olopade etal., 1992; Rowley and Diaz, 1992), suggesting a role for the tek locusin oncogenesis.

The novel gene products of the invention were identified as mousereceptor tyrosine kinase protein based on the structural homology of theprotein to the known mouse and human receptor tyrosine kinases. Thededuced amino acid sequence of Tek protein predicts that it encodes aputative receptor tyrosine kinase that contains a 21 amino acid kinaseinsert and which is most closely related in its catalytic domain toFGFR1 (mouse fibroblast growth factor) and the product of the retproto-oncogene.

Northern blot hybridization analysis of RNA from 12.5 day embryonicheart using the 1.6 kb cDNA as probe suggested that the tek locus givesrise to at least 4 different transcripts of approximately 4.5, 2.7, 2.2,and 0.8 kb. Differential splicing of primary transcripts is known tooccur for several genes encoding RTKs, including met (Rodrigues, G. A.,Naujokas, M. A. & Park, M. (1991), Mol.Cell.Biol., 11, 2962-2970), trkB(Middlemas, D. S., Lindberg, R. A. & Hunter, T. (1991), Mol.Cell.Biol.,11, 143-153), ret (Tahira, T., Ishizaka, Y., Itoh, F., Sugimura, T. &Nagao, M. (1990), Oncogene, 5, 97-102), and flg (Reid et al., 1990,Proc.Natl.Acad.Sci., 87,1596-1600; Bernard, O., Li, M. & Reid, H. H.(1991), Proc.Natl.Acad Sci.USA, 88, 7625-7629; Eisemann, A., Ahn, J. A.,Graziani, G., Tronick, S. R. & Ron, D. (1991), Oncogene, 6, 1195-1202;Fujita, H., Ohta, M., Kawasaki, T. & Itoh, N. (1991), Biochem. Biophis.Res. Comm., 174, 946-951; Meng, B. & Reid, H. H. (1991),Proc.Natl.Acad.Sci., 7625-7629), favoring the possibility that at leastsome of the smaller transcripts hybridizing with the tek cDNA aredifferentially spliced. The 4.5 kb tek transcript is of the appropriatesize to encode a molecule with an extensive extracellular domain. Incontrast, the smallest transcript, at 0.8 kb, is sufficient to encodeonly a significantly truncated version of the protein. Since thistranscript was detected with a probe comprised entirely of sequencesfrom the catalytic domain and 3' untranslated region, it is possiblethat the 0.8 kb message codes for an isoform completely lacking anextracellular domain. Truncated molecules of this type have recentlybeen shown to be encoded by the trkB gene in rats (Middlemas et al.,1991, Mol.Cell.Biol., 11, 143-153) and by pdgfb in murine ES cells (Vu,T. H., Martin, G. R., Lee, P., Mark, D., Wang, A. & Williams, L. T.(1989), Mol.Cell.Biol., 9, 4563-4567). These small isoforms may act ascatalytically deregulated molecules during periods of rapid growth(Middlemas et al., 1991). The detection of multiple tek transcripts mayindicate potential differential expression of different tek isoformsduring embryogenes is.

Overlapping cDNAs from tek hybridizing clones were used to assemble a4177 nucleotide contiguous cDNA (FIG. 11B and SEQ ID NO: 5). Thesequence of this cDNA predicts a 1122-residue protein having severalstructural motifs that distinguish it from other receptor tyrosinekinases. In particular the Tek tyrosine kinase protein has anextracellular domain within which three distinct types of structuralmotifs can be identified, including immunoglobulin-like loops betweenresidues 19 and 209 and 344 and 467 (FIG. 11B and SEQ ID NO: 6). The twoimmunoglobulin-like loops are separated from one another by three tandemcysteine-rich epidermal growth factor (EGF)-like repeats (SEQ IDNO:17-19) that show homology to similar motifs found in othercell-surface proteins, such as Tie (SEQ ID NO:20-22) and Notch (SEQ IDNO:24) (FIG. 12A). Moreover, the second immunoglobulin-like loop isfollowed by three regions (SEQ ID NO:25, 27 and 29) showing homology tofibronectin type III (FNIII) repeats found in polypeptides such asDrosophila leukocyte common antigen-related molecule (DLAR) (SEQ IDNO:33) and fibronectin (FIG. 12B). The extracellular domain of Tekreceptor tyrosine kinase protein represents a composite of threedifferent structural motifs that are usually not found collectivelywithin a single receptor tyrosine kinase.

It is likely that the unusual structure of the Tek receptor tyrosinekinase protein reflects some aspect of its role in endothelial cellbiology. In addition to playing potential roles in regulatingendothelial cell proliferation and differentiation, the complexstructure of the Tek receptor tyrosine kinase protein extracellulardomain likely also plays a role in guiding the proper patterning ofendothelial cells during blood vessel formation, both in the embryo andin the adult.

Tie, a receptor tyrosine kinase protein expressed in cells of theendothelial lineage (Partenan et al, 1992, Mol. Cell Biol. 12:1698-1707)shows a similar juxtaposition of structural motifs within theextracellular domain as Tek receptor tyrosine kinase protein. Despitethe structural homology between Tek and Tie proteins, these twomolecules show only modest sequence similarity in their extracellulardomains (FIGS. 12A and 12B), suggesting that they interact with distinctligands. In addition, Tek and Tie proteins are more divergent withintheir carboxy terminal tails and kinase insert regions than in theirATP-binding and phosphotransferase domains, suggesting that these tworeceptors likely utilize non-identical signalling pathways.

A 140-kDa protein was specifically precipitated from a cell linetransfected with tek cDNA (FIG. 15A). Moreover, this 140 kDa proteincould be detected immunologically by Western analysis (FIG. 15B, lane 2)and its immunoprecipitation could be competed by a GST fusion proteincontaining the 43-residue carboxy terminal segment to which the antibodywas raised (FIG. 15A, lane 3). The apparent size of the encoded Tekreceptor tyrosine kinase protein, 140 kDa, is approximately 20 kDagreater than that predicted by the deduced amino acid sequence (126kDa). The larger size of the detected protein indicates that Tekreceptor tyrosine kinase protein may be a glycosylated cell surfaceprotein.

Cell lysates prepared from umbilical vein, Py4-1 cells, and Day 13.5embryonic heart all contained a 140 kDa protein that reactedspecifically with Tek antibody and which comigrated with the speciesdetected in transfected COS cells. Taken together, the results indicatethat the 4.2 Kb tek cDNA contains the complete coding information forthe native Tek receptor tyrosine kinase protein.

The tek cDNA encodes a 140 kDa protein which comigrates with thepolypeptide specifically detected by Tek antibody in both culturedendothelial cells (Py4-1) and highly vascularized embryonic tissues(heart and umbilical vein). The Tek receptor tyrosine kinase proteincytoplasmic domain expressed in E. coli was shown to react withphosphotyrosine receptor tyrosine kinase protein antibodies.

The DNA sequence and deduced amino acid sequence of tek are shown in SEQID NOS: 1 and 2 and FIG. 1, and in SEQ ID NOS: 5 and 6 and FIG. 11B. TheDNA sequence and deduced amino acid sequence of a 1601 bp segment areshown in SEQ ID NOS: 3 and 4 and in FIG. 2. The DNA and deduced aminoacid sequence of tek shown in FIG. 1 and SEQ ID NOS: 1 and 2 are thesame as those shown in FIG. 11B and SEQ ID NOS: 5 and 6, with theexception that FIG. 11B and SEQ ID NOS: 5 and 6 have an additional shortsegment of 12 nucleotides, (coding for the amino acids Phe, Gln, Asp,Val) commencing at nucleotide number 2592. This short segment is alsoshown in FIG. 2 and SEQ ID NO: 3 commencing at nucleotide number 7.

It will be appreciated that the invention includes nucleotide or aminoacid sequences which have substantial sequence homology with thenucleotide and amino acid sequences shown in SEQ ID NOS:1-6 and in FIGS.1, 2 and 11B. The term "sequences having substantial sequence homology"means those nucleotide and amino acid sequences which have slight orinconsequential sequence variations from the sequences disclosed inFIGS. 1, 2 and 11B and SEQ ID NOS: 1-6, i.e. the homologous sequencesfunction in substantially the same manner to produce substantially thesame polypeptides as the actual sequences. The variations may beattributable to local mutations or structural modifications.

Sequences having substantial homology include nucleic acid sequenceswhich encode proteins having at least 95% sequence homology with theamino acid sequences as shown in SEQ ID NOS: 2, 4 and 6 or portionsthereof; and nucleic acid sequences having at least 85% homology,preferably at least 90% with the nucleic acid sequences as shown in SEQID NOS: 1 and 5 or fragments thereof. An example of such a sequenceincludes the sequence encoding Tek receptor tyrosine kinase protein inhumans and in other mammals.

Sequences having substantial homology also include fragments of thenucleic acid sequences of the invention having at least 18 bases whichwill hybridize to the nucleic acid sequences under stringent conditions.Stringent hybridization conditions are those which are stringent enoughto provide specificity, reduce the number of mismatches and yet aresufficiently flexible to allow formation of stable hybrids at anacceptable rate. Such conditions are known to those skilled in the artand are described, for example, in Sambrook, et al, (1989, MolecularCloning, A Laboratory Manual, Cold Spring Harbor). By way of exampleonly, stringent hybridization with short nucleotides may be carried outat 5-10° below the T_(m) using high concentrations of probe such as0.01-1.0 pmole/ml.

The invention also provides amino acid sequences having substantialsequence homology with the amino acid sequence shown in SEQ ID NO: 2, 4or 6. Substantially homologous sequences include sequences having atleast 95% sequence homology. Peptides which are unique to the receptortyrosine kinase protein of the invention are also contemplated,preferably peptides having at least 10 amino acids.

It will also be appreciated that a double stranded nucleotide sequencecomprising a nucleic acid molecule of the invention or anoligonucleotide fragment thereof, hydrogen bonded to a complementarynucleotide base sequence, an RNA made by transcription of this doublestranded nucleotide sequence, and an antisense strand of the nucleicacid molecule of the invention or an oligonucleotide fragment of thenucleic acid molecule, are contemplated within the scope of theinvention.

The sequence of the nucleic acid molecule of the invention or a fragmentthereof, may be inverted relative to its normal presentation fortranscription to produce antisense nucleic acid molecules. The antisensenucleic acid molecules may be constructed using chemical synthesis andenzymatic ligation reactions using procedures known in the art.

A number of unique restriction sequences for restriction enzymes areincorporated in the nucleic acid sequences identified in SEQ ID NOS: 1,3 and 5 and in FIGS. 1, 2 and 11B and these provide access to nucleotidesequences which code for polypeptides unique to the receptor tyrosinekinase protein of the invention. DNA sequences unique to the receptortyrosine kinase protein of the invention or isoforms thereof, can alsobe constructed by chemical synthesis and enzymatic ligation reactionscarried out by procedures known in the art.

The present invention includes conjugates of the receptor tyrosinekinase protein of the invention. For example, the receptor tyrosinekinase protein or parts thereof may be conjugated with selected proteinsto produce fusion proteins. Examples of proteins which may be selectedinclude lymphokines such as gamma interferon, tumor necrosis factor,IL-1, IL-2, IL-3, Il-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,GM-CSF, CSF-1 and G-CSF. Particularly preferred molecules include the Fcportion of immunoglobulin molecules.

II. Expression Pattern of the Receptor Tyrosine Kinase Protein of theInvention

In the adult and all stages of embryonic development examined, tekexpression was primarily restricted to cells of the endothelial lineage.Tek transcripts have also been found by the present inventors in themesoderm of the amnion of developing embryos. The amnion is comprised oftwo cell layers, one mesodermal and the other ectodermal in origin. Thismembrane shares several features with the endothelial lining of bloodvessels, such as having an epithelial-like morphology and therequirement to contain fluid within an enclosed cavity. Thus, thistissue may utilize Tek receptor tyrosine kinase protein to accomplishthis.

Specifically, in situ hybridization analysis of adult tissues, as wellas sectioned and whole mount embryos, showed that tek is specificallyexpressed in the endocardium, the leptomeninges and the endotheliallining of the vasculature from the earliest stages of their development.Moreover, examination of the morphology of tek-expressing cells, andstaging of tek expression relative to that of the endothelial cellmarker von Willebrand factor, revealed that tek is expressed prior tovon Willebrand factor and appears to mark the embryonic progenitors ofmature endothelial cells. Thus, tek encodes a novel putative receptortyrosine kinase that may be critically involved in the determinationand/or maintenance of cells of the endothelial lineage.

Overall, the pattern of expression observed in sectioned and whole mountmouse embryos was similar to that described previously for quail embryosstained with a monoclonal antibody specific for cells of the endotheliallineage (Pardanaud, L., Altmann, C., Kitos, P., Dieterlen-Lievre, F. &Buck, C. A. (1987). Development, 100, 339-349; Coffin, J. D. & Poole, T.J. (1988). Development, 102, 735-748). Thus, it is likely thatorchestration of vascularization in the two vertebrate species is verysimilar. Studies on cell lineage relations carried out primarily in thechick (Noden, D. M. (1989), Am.Rev.Respir.Dis., 140, 1097-1103, andNoden, D. M. (1990), Ann. N.Y. Acad. Sci., 1, 236-249; O'Brien, S. J.Genetic Maps, Locus Maps of Complex Genomes. Cold Spring HarborLaboratory Press, 1990) have established that endothelial cells arederived from angioblasts, which migrate from mesoderm and populate theembryo with precursor cells that eventually contribute to the formationof the intraembryonic blood vessels.

FIG. 10 shows the hierarchy of the endothelial cell lineage. Horizontalbars denote the relationship between cellular determination and onset ofexpression of tek and von Willebrand factor within the lineage (adaptedfrom (Wagner, R. C. (1980). Adv.Microcirc., 9, 45-75). In the yolk sac,angioblasts are thought to originate from hemangioblasts, ill-definedcells of mesenchymal origin that are also believed to give rise toprimitive blood cells in the developing blood islets. In the embryo, onthe other hand, angioblasts are thought to arise directly from cells ofthe mesenchymal anlage (Wagner, 1980).

Several cell lines of endothelial origin were also examined forexpression of tek and of Flk-1. Flk-1 encodes a receptor tyrosine kinaseprotein which is expressed in cells of the endothelial lineage. Tek andFlk-1 were differentially expressed in endothelial cell lines (FIG. 14),suggesting that tek and Flk-1 are differentially regulated.

The present inventors' work suggested that tek is expressed in thepresumptive precursors of endothelial cells, the angioblasts. First, tekexpression was detected in both von Willebrand factor-positive cells aswell as cells that appear to be progenitors of endothelial cells.Second, tek expression was observed in cells of non-endothelialmorphology that in the avian system have been identified previously asangioblasts. It may also be significant that in the 8.5 day embryo, tekexpression was identified in cells extending beneath the ventral surfaceof somites (FIG. 6, J). Analysis of serial sections revealed that someof these tek-expressing cells were actually contiguous with the somites.These cells may correspond to those described by Beddington, R. S. P. &Martin, P. (1989), Mol.Cell.Med., 6, 263-274 who showed in mouse tissuetransplantation studies that lacZ-expressing somite tissue, while devoidof endothelial cells prior to transplantation, possess cells capable ofmigrating and contributing to the host vasculature. Taken together, thepresent inventors' work suggests that tek expression may constitute oneof the earliest mammalian endothelial cell lineage markers described todate.

The restricted expression of tek, imposes constraints on the cellularrange of activity of the putative Tek receptor tyrosine kinase proteinligand, and suggests that the tek locus probably plays unique andimportant roles in the determination, migration, or proliferation ofcells of the endothelial lineage.

Tek expression is very low in adults. However, it is likely thatexpression will be upregulated upon induction of angiogenesis.Accordingly, tek likely plays a role in angiogenesis, for example intumor growth, in mature animals in addition to its role duringdevelopment.

III. Preparation of Nucleic Acid Molecules and Proteins of the Invention

As hereinbefore mentioned, the present inventors have identified andsequenced a cDNA sequence encoding a novel receptor tyrosine kinaseprotein designated Tek.

Nucleic acid molecules of the present invention encoding the novelreceptor tyrosine kinase protein of the present invention, or related,or analogous sequences, may be isolated and sequenced, for example, bysynthesizing cDNAs from embryonic heart RNA by RT-PCR using degenerateoligonucleotide primers which amplify tyrosine kinase sequences such asthe two degenerate tyrosine kinase oligonucleotide primers described byWilks, A. F. ((1989) Proc.Natl.Acad.Sci., 86, 1603-1607) and analysingthe sequences of the clones obtained following amplification. Nucleicacid molecules of the present invention, or fragments thereof, encodingthe novel receptor tyrosine kinase protein of the present invention, orparts thereof, may also be constructed by chemical synthesis andenzymatic ligation reactions using procedures known in the art.

The nucleic acid molecules of the present invention having a sequencewhich codes for the receptor tyrosine kinase protein of the invention,or an oligonucleotide fragment of the nucleic acid molecules may beincorporated in a known manner into a recombinant molecule which ensuresgood expression of the protein or part thereof. In general, arecombinant molecule of the invention contains a nucleic acid molecule,or an oligonucleotide fragment thereof, of the invention and anexpression control sequence operatively linked to the nucleic acidmolecule or oligonucleotide fragment. A nucleic acid molecule of theinvention or an oligonucleotide fragment thereof, may be incorporatedinto a plasmid vector, for example, pECE. Suitable regulatory elementsmay be derived from a variety of sources, including bacterial, fungal,viral, mammalian, or insect genes. Selection of appropriate regulatoryelements is dependent on the host cell chosen, and may be readilyaccomplished by one of ordinary skill in the art. Examples of regulatoryelements include: a transcriptional promoter and enhancer or RNApolymerase binding sequence, a ribosomal binding sequence, including atranslation initiation signal. Additionally, depending on the host cellchosen and the vector employed, other genetic elements, such as anorigin of replication, additional DNA restriction sites, enhancers,sequences conferring inducibility of transcription, and selectablemarkers, may be incorporated into the expression vector.

The Tek receptor tyrosine kinase protein or isoforms or parts thereof,may be obtained by expression in a suitable host cell using techniquesknown in the art. Suitable host cells include prokaryotic or eukaryoticorganisms or cell lines, for example, yeast, E. coli and mouse NIH 3Bcells may be used as host cells. The protein or parts thereof may beprepared by chemical synthesis using techniques well known in thechemistry of proteins such as solid phase synthesis (Merrifield, 1964,J. Am. Chem. Assoc. 85:2149-2154) or synthesis in homogenous solution(Houbenweyl, 1987, Methods of Organic Chemistry, ed. E. Wansch, Vol. 15I and II, Thieme, Stuttgart).

DNA sequences encoding Tek receptor tyrosine kinase protein, or a partthereof, may be expressed by a wide variety of prokaryotic andeukaryotic host cells, including bacterial, mammalian, yeast or otherfungi, viral, plant, or insect cells. Methods for transforming ortransfecting such cells to express foreign DNA are well known in the art(see, e.g., Itakura et al., U.S. Pat. No. 4,704,362; Hinnen et al., PNASUSA 75:1929-1933, 1978; Murray et al., U.S. Pat. No. 4,801,542; Upshallet al., U.S. Pat. No. 4,935,349; Hagen et al., U.S. Pat. No. 4,784,950;Axel et al., U.S. Pat. No. 4,399,216; Goeddel et al., U.S. Pat. No.4,766,075; and Sambrook et al. Molecular Cloning A Laboratory Manual,2nd edition, Cold Spring Harbor Laboratory Press, 1989, all of which areincorporated herein by reference).

Bacterial host cells suitable for carrying out the present inventioninclude E. coli, B. subtilis, Salmonella typhimurium, and variousspecies within the genus' Pseudomonas, Streptomyces, and Staphylococcus,as : well as many other bacterial species well known to one of ordinaryskill in the art. Representative examples of bacterial host cellsinclude DH5α (Stratagene, LaJolla, Calif.), JM109 ATCC No. 53323, HB101ATCC No. 33694, and MN294.

Bacterial expression vectors preferably comprise a promoter whichfunctions in the host cell, one or more selectable phenotypic markers,and a bacterial origin of replication. Representative promoters includethe β-lactamase (penicillinase) and lactose promoter system (see Changet al., Nature 275:615, 1978), the trp promoter (Nichols and Yanofsky,Meth in Enzymology 101:155, 1983) and the tac promoter (Russell et al.,Gene 20: 231, 1982). Representative selectable markers include variousantibiotic resistance markers such as the kanamycin or ampicillinresistance genes. Many plasmids suitable for transforming host cells arewell known in the art, including among others, pBR322 (see Bolivar etal., Gene 2:9S, 1977), the pUC plasmids pUC18, pUC19, pUC118, pUC119(see Messing, Meth in Enzymology 101:20-77, 1983 and Vieira and Messing,Gene 19:259-268, 1982), and pNH8A, pNH16a, pNH18a, and Bluescript M13(Stratagene, La Jolla, Calif.).

Yeast and fungi host cells suitable for carrying out the presentinvention include, among others Saccharomyces cerevisiae, the generaPichia or Kluyveromyces and various species of the genus Aspergillus.Suitable expression vectors for yeast and fungi include, among others,YC.sub.ρ 50 (ATCC No. 37419) for yeast, and the amdS cloning vector pV3(Turnbull, Bio/Technology 7:169, 1989). Protocols for the transformationof yeast are also well known to those of ordinary skill in the art. Forexample, transformation may be readily accomplished either bypreparation of spheroplasts of yeast with DNA (see Hinnen et al., PNASUSA 75:1929, 1978) or by treatment with alkaline salts such as LiCl (seeItoh et al., J. Bacteriology 153:163, 1983). Transformation of fungi mayalso be carried out using polyethylene glycol as described by Cullen etal. (Bio/Technology 5:369, 1987).

Mammalian cells suitable for carrying out the present invention include,among others: COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g., ATCC No.CRL 6281), CHO (ATCC No. CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCCNo. 1573) and NS-1 cells. Suitable expression vectors for directingexpression in mammalian cells generally include a promoter, as well asother transcriptional and translational control sequences. Commonpromoters include SV40, MMTV, metallothionein-1, adenovirus Ela, CMV,immediate early, immunoglobulin heavy chain promoter and enhancer, andRSV-LTR. Protocols for the transfection of mammalian cells are wellknown to those of ordinary skill in the art. Representative methodsinclude calcium phosphate mediated electroporation, retroviral, andprotoplast fusion-mediated transfection (see Sambrook et al., supra).

Given the teachings provided herein, promoters, terminators, and methodsfor introducing expression vectors of an appropriate type into plant,avian, and insect cells may also be readily accomplished. For example,within one embodiment, tek or derivatives thereof may be expressed fromplant cells (see Sinkar et al., J. Biosci (Bangalore) 11:47-58, 1987,which reviews the use of Agrobacterium rhizogenes vectors; see alsoZambryski et al., Genetic Engineering, Principles and Methods,Hollaender and Setlow (eds.), Vol. VI, pp. 253-278, Plenum Press, NewYork, 1984, which describes the use of expression vectors for plantcells, including, among others, pAS2022, pAS2023, and pAS2034).

Tek receptor tyrosine kinase protein may be prepared by culturing thehost/vector systems described above, in order to express the recombinantTek receptor tyrosine kinase protein.

Conjugates of Tek receptor tyrosine kinase protein of the invention, orparts thereof, with other molecules, such as proteins or polypeptides,may be prepared. This may be accomplished, for example, by the synthesisof N-terminal or C-terminal fusion proteins. Thus, fusion proteins maybe prepared by fusing, through recombinant techniques, the N-terminal orC-terminal of Tek receptor tyrosine kinase protein or parts thereof, andthe sequence of a selected protein with a desired biological function.The resultant fusion proteins contain Tek receptor tyrosine kinaseprotein or a portion thereof fused to the selected protein. Examples ofproteins which may be selected to prepare fusion proteins includelymphokines such as gamma interferon, tumor necrosis factor, IL-1, IL-2,IL-3, Il-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, GM-CSF, CSF-1and G-CSF. Particularly preferred molecules include the Fc portion ofimmunoglobulin molecules.

Sequences which encode the above-described molecules may generally beobtained from a variety of sources, including for example, depositorieswhich contain plasmids encoding sequences including the American TypeCulture Collection (ATCC, Rockville Md.), and the British BiotechnologyLimited (Cowley, Oxford England). Examples of such plasmids include BBG12 (containing the GM-CSF gene coding for the mature protein of 127amino acids), BBG 6 (which contains sequences encoding gammainterferon), ATCC No. 39656 (which contains sequences encoding TNF),ATCC No. 20663 (which contains sequences encoding alpha interferon,)ATCC Nos. 31902 and 39517 (which contains sequences encoding betainterferon), ATCC No. 67024 (which contains a sequence which encodesInterleukin-1β), ATCC Nos. 39405, 39452, 39516, 39626 and 39673 (whichcontains sequences encoding Interleukin-2), ATCC Nos. 59399, 59398, and67326 (which contain sequences encoding Interleukin-3), ATCC Nos. 57592(which contains sequences encoding Interleukin-4). ATCC Nos. 59394 and59395 (which contain sequences encoding Interleukin-5), and ATCC No.67153 (which contains sequences encoding Interleukin-6.

Within a particularly preferred embodiment of the invention, tek iscloned into an expression vector as a fusion gene with the constantregion of human immunoglobulin γ1. Briefly, the expression vectorspNUTΔGH and pVL1393 are prepared for cloning by digestion with SmaIfollowed by dephosphorylation by calf intestinal alkaline phosphatase.The linear product is isolated after agarose gel electrophoresis. Thetek genes are then generated by polymerase chain reaction using thecloned tek cDNA as a template. In particular, the Tek fusion protein issynthesized from the extracellular domain of Tek receptor tyrosinekinase protein (amino acids 19 to 744, SEQ ID NO: 6 and FIG. 11B).

The constant region of an immunoglobulin, such as human γ1 gene may beprepared, for example, from pUCB7Ig monomer. Briefly, the C_(H) gene isisolated by digestion with XbaI which cuts at the 3' end of the genefollowed by treatment with E. coli DNA polymerase I in the presence ofall four dNTPs in order to create a blunt end. The plasmid is thendigested with BclI which cuts at the 5' end of the gene. The fragmentcontaining the heavy chain gene is isolated after electrophoresis in anagarose gel.

The fusion tek amplified fragment is inserted into each prepared vectoralong with the heavy chain fragment. Orientation of the resultingplasmids is determined by PCR with one priming oligo which anneals tovector sequence and the other priming oligo which anneals to the insertsequence. Alternatively, appropriate restriction digests can beperformed to verify the orientation. The sequence of the fusiontek/immunoglobulin constant region gene can be verified by DNAsequencing.

Phosphorylated receptor tyrosine kinase proteins of the invention, orparts thereof, may be prepared using the method described in Reedijk etal. The EMBO Journal 11(4):1365, 1992. For example, tyrosinephosphorylation may be induced by infecting bacteria harbouring aplasmid containing a nucleotide sequence of the invention or fragmentthereof, with a λgt11 bacteriophage encoding the cytoplamic domain ofthe Elk tyrosine kinase. Bacteria containing the plasmid andbacteriophage as a lysogen are isolated. Following induction of thelysogen, the expressed receptor protein becomes phosphorylated.

Alternatively, tek may be expressed in non-human transgenic animals suchas, rats, rabbits, sheep and pigs (see Hammer et al. (Nature315:680-683, 1985), Palmiter et al. (Science 222:809-814, 1983),Brinster et al. (Proc Natl. Acad. Sci USA 82:44384442, 1985), Palmiterand Brinster (Cell. 41:343-345, 1985) and U.S. Pat. No. 4,736,866).

IV. Utility of the Nucleic Acid Molecules and Proteins of the Invention

The nucleic acid molecules of the invention or oligonucleotide fragmentsthereof, allow those skilled in the art to construct nucleotide probesfor use in the detection of nucleotide sequences in biologicalmaterials. A nucleotide probe may be labelled with a radioactive labelwhich provides for an adequate signal and has sufficient half-life suchas ³² p, ³ H, ¹⁴ C or the like. Other labels which may be used includeantigens that are recognized by a specific labelled antibody,fluorescent compounds, enzymes, antibodies specific for a labelledantigen, and chemiluminescense. An appropriate label may be selectedhaving regard to the rate of hybridization and binding of the probe tothe nucleotide to be detected and the amount of nucleotide available forhybridization. Labelled probes may be hybridized to nucleic acids onsolid supports such as nitrocellulose filters or nylon membranes asgenerally described in Sambrook et al, 1989, Molecular Cloning, ALaboratory Manual (2nd Edition). The nucleotide probes may be used todetect genes, preferably in human cells, that encode proteins relatedto, or analogous to, the novel receptor tyrosine kinase protein of theinvention.

The receptor tyrosine kinase protein of the invention or parts thereof,for example amino acids of the extracellular domain, carboxy terminaltail or catalytic domain, may be used to prepare monoclonal orpolyclonal antibodies. Antibodies having specificity for Tek receptortyrosine kinase protein may also be raised from fusion proteins createdby expressing trpE-Tek fusion proteins in bacteria as described above.

Within the context of the present invention, antibodies are understoodto include monoclonal antibodies, polyclonal antibodies, antibodyfragments (e.g., Fab, and F(ab')₂ and recombinantly produced bindingpartners. Antibodies are understood to be reactive against Tek receptortyrosine kinase protein if they bind with a K_(a) of greater than orequal to 10⁻⁷ M. As will be appreciated by one of ordinary skill in theart, antibodies may be developed which not only bind to Tek protein, butwhich bind to a ligand of Tek protein, and which also block thebiological activity of Tek protein. Such antibodies will be useful inthe diagnosis and treatment of developmental disorders of endothelialcell growth, angiogenesis, vascularization, wound healing andtumorigenesis.

Conventional methods can be used to prepare the antibodies as discussedin more detail below. As to the details relating to the preparation ofmonoclonal antibodies reference can be made to Goding, J. W., MonoclonalAntibodies: Principles and Practice, 2nd Ed., Academic Press, London,1986; U.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993which are incorporated herein by reference; see also MonoclonalAntibodies, Hybridomas: A New Dimension in Biological Analyses, PlenumPress, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: ALaboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor LaboratoryPress, 1988, which are also incorporated herein by reference).

Other techniques may also be utilized to construct monoclonal antibodies(see William D. Huse et al., "Generation of a Large CombinationalLibrary of the Immunoglobulin Repertoire in Phage Lambda," Science246:1275-1281, December 1989; see also L. Sastry et al., "Cloning of theImmunological Repertoire in Escherichia coli for Generation ofMonoclonal Catalytic Antibodies: Construction of a Heavy Chain VariableRegion-Specific cDNA Library," Proc Natl. Acad. Sci USA 86:5728-5732,August 1989; see also Michelle Alting-Mees et al., "Monoclonal AntibodyExpression Libraries: A Rapid Alternative to Hybridomas," Strategies inMolecular Biology 3:1-9, January 1990; these references, which are alsoincorporated herein by reference, describe a commercial system availablefrom Stratacyte, La Jolla, Calif., which enables the production ofantibodies through recombinant techniques).

Binding partners may also be constructed utilizing recombinant DNAtechniques to incorporate the variable regions of a gene which encodes aspecifically binding antibody. Within one embodiment, the genes whichencode the variable region from a hybridoma producing a monoclonalantibody of interest are amplified using nucleotide primers for thevariable region. These primers may be synthesized by one of ordinaryskill in the art, or may be purchased from commercially availablesources. Stratacyte (La Jolla, Calif.) sells primers for mouse and humanvariable regions including, among others, primers for V_(Ha), V_(Hb),V_(Hc), V_(Hd), C_(H1), V_(L) and C_(L) regions. These primers may beutilized to amplify heavy or light chain variable regions, which maythen be inserted into vectors such as ImmunoZAP™ H or ImmunoZAP™ L(Stratacyte), respectively. These vectors may then be introduced into E.coli for expression. Utilizing these techniques, large amounts of asingle-chain protein containing a fusion of the VH and VL domains may beproduced (See Bird et al., Science 242:423-426, 1988). In addition, suchtechniques may be utilized to change a "murine" antibody to a "human"antibody, without altering the binding specificity of the antibody.

Once suitable antibodies or binding partners have been obtained, theymay be isolated or purified by many techniques well known to those ofordinary skill in the art (see Antibodies: A Laboratory Manual, Harlowand Lane (eds.), Cold Spring Harbor Laboratory Press, 1988). Suitabletechniques include peptide or protein affinity columns, HPLC or RP-HPLC,purification on protein A or protein G columns, or any combination ofthese techniques.

The polyclonal or monoclonal antibodies may be used to detect thereceptor tyrosine kinase protein of the invention in various biologicalmaterials, for example they may be used in an Elisa, radioimmunoassay orhistochemical tests. Thus, the antibodies may be used to quantify theamount of a receptor tyrosine kinase protein of the invention in asample in order to determine its role in particular cellular events orpathological states.

In particular, the polyclonal and monoclonal antibodies of the inventionmay be used in immunohistochemical analyses, for example, at thecellular and sub-subcellular level, to detect the novel receptortyrosine kinase protein of the invention, to localise it to particularcells and tissues and to specific subcellular locations, and toquantitate the level of expression.

Cytochemical techniques known in the art for localizing antigens usinglight and electron microscopy may be used to detect the novel tyrosinekinase of the invention. Generally, an antibody of the invention may belabelled with a detectable substance and the novel receptor tyrosinekinase of the invention may be localised in tissue based upon thepresence of the detectable substance. Examples of detectable substancesinclude various enzymes, fluorescent materials, luminescent materialsand radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, biotin, alkaline phosphatase, β-galactosidase,or acetylcholinesterase; examples of suitable fluorescent materialsinclude umbelliferone, fluorescein, fluorescein isothiocyanate,rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride orphycoerythrin; an example of a luminescent material includes luminol;and examples of suitable radioactive materials include radioactiveiodine I¹²⁵, I¹³¹ or tritium. Antibodies may also be coupled to electrondense substances, such as ferritin or colloidal gold, which are readilyvisualised by electron microscopy.

Radioactive labelled materials may be prepared by radiolabeling with ¹²⁵I by the chloramine-T method (Greenwood et al, Biochem. J. 89:114,1963), the lactoperoxidase method (Marchalonis et al, Biochem. J.124:921, 1971), the Bolton-Hunter method (Bolton and Hunter, Biochem. J.133:529, 1973 and Bolton Review 18, Amersham International Limited,Buckinghamshire, England, 1977), the iodogen method (Fraker and Speck,Biochem. Biophys. Res. Commun. 80:849, 1978), the Iodo-beads method(Markwell Anal. Biochem. 125:427, 1982) or with tritium by reductivemethylation (Tack et al., J. Biol. Chem. 255:8842, 1980).

Known coupling methods (for example Wilson and Nakane, in"Immunofluorescence and Related Staining Techniques", W. Knapp et al,eds, p. 215, Elsevier/North-Holland, Amsterdam & New York, 1978; P.Tijssen and E. Kurstak, Anal. Biochem. 136:451, 1984) may be used toprepare enzyme labelled materials. Fluorescent labelled materials may beprepared by reacting the material with umbelliferone, fluorescein,fluorescein isothiocyanate, dichlorotriazinylamine fluorescein, dansylchloride, derivatives of rhodamine such as tetramethyl rhodamineisothiocyanate, or phycoerythrin.

Indirect methods may also be employed in which the primaryantigen-antibody reaction is amplified by the introduction of a secondantibody, having specificity for the antibody reactive against the noveltyrosine kinase of the invention. By way of example, if the antibodyhaving specificity against the novel tyrosine kinase protein of theinvention is a rabbit IgG antibody, the second antibody may be goatanti-rabbit gamma-globulin labelled with a detectable substance asdescribed herein.

Where a radioactive label is used as a detectable substance, the noveltyrosine kinase of the invention may be localized by radioautography.The results of radioautography may be quantitated by determining thedensity of particles in the radioautographs by various optical methods,or by counting the grains.

As discussed above, the expression patterns found for the novel tyrosinekinase of the invention indicate that it plays unique and importantroles in angiogenesis, cardiogenesis and tumorigenesis. Therefore, theabove described methods for detecting nucleic acid molecules andfragments thereof and Tek protein and parts thereof, can be used tomonitor angiogenesis, cardiogenesis and tumorigenesis by detecting andlocalizing the novel tyrosine kinase protein of the invention.

It would also be apparent to one skilled in the art that the abovedescribed methods may be used to study the developmental expression ofTek and, accordingly, will provide further insight into the role of Tekprotein in angiogenesis, cardiogenesis and tumorigenesis.

The finding of a novel receptor tyrosine kinase which is only expressedin cells of the endothelial lineage permits the identification ofsubstances such as ligands, which may affect angiogenesis and/ormaintenance of cells of the endothelial lineage and which may play arole in tumorigenesis. Therefore, in accordance with a method of theinvention ligands, and natural and synthetic derivatives of suchligands, which are capable of binding to, and in some cases activatingthe receptor tyrosine kinase protein of the invention, isoforms thereof,or part of the protein may be identified. The method involves reactingthe novel receptor kinase protein of the invention, isoforms thereof, orpart of the protein with at least one ligand which potentially iscapable of binding to the protein, isoform or part of the protein, underconditions which permit the formation of ligand-receptor proteincomplexes, and assaying for ligand-receptor protein complexes, for freeligand or for non-complexed proteins or for activation of the receptortyrosine kinase.

The ligand-receptor protein complexes, free ligand or non-complexedproteins receptor-ligand complex, may be isolated by conventionalisolation techniques, for example, salting out, chromatography,electrophoresis, gel filtration, fractionation, absorption,polyacrylamide gel electrophoresis, agglutination, or combinationsthereof. To facilitate the assay of the components, antibody against thereceptor protein or the ligand, or a labelled receptor protein, or alabelled ligand may be utilized. Antibodies, receptor protein, orsubstance may be labelled with a detectable substance as describedabove.

The receptor tyrosine kinase protein, isoforms or parts thereof, orligand used in the method of the invention may be insolubilized. Forexample, the receptor protein or ligand may be bound to a suitablecarrier. Examples of suitable carriers are agarose, cellulose, dextran,Sephadex, Sepharose, carboxymethyl cellulose polystyrene, filter paper,ion-exchange resin, plastic film, plastic tube, glass beads,polyamine-methyl vinyl-ether-maleic acid copolymer, amino acidcopolymer, ethylene-maleic acid copolymer, nylon, silk, etc. The carriermay be in the shape of, for example, a tube, test plate, beads, disc,sphere etc. Insolubilized receptor tyrosine kinase protein or ligandthereof will include receptor tyrosine kinase protein or ligand thereofexpressed on the surface of a cell.

The insolubilized receptor tyrosine kinase protein or ligand may beprepared by reacting the material with a suitable insoluble carrierusing known chemical or physical methods, for example, cyanogen bromidecoupling.

Conditions which permit the formation of ligand-receptor proteincomplexes may be selected having regard to factors such as the natureand amounts of the ligand and the receptor protein.

The receptor tyrosine kinase protein, parts thereof, or substances mayalso be expressed on the surface of a cell using the methods describedherein.

In a preferred embodiment of the method, ligands are identified whichare capable of binding to and activating the novel receptor tyrosinekinase protein of the invention. In this method the ligands which bindto and activate the novel receptor tyrosine kinase protein of theinvention are identified by assaying for protein tyrosine kinaseactivity i.e. by assaying for phosphorylation of the tyrosine residuesof the receptor.

Protein tyrosine kinase activity may be assayed using known techniquessuch as those using antiphosphotyrosine antibodies and labelledphosphorous. For example, immunoblots of the complexes may be analyzedby autoradiography (³² P-labelled samples) or may be blocked and probedwith antiphosphotyrosine antibodies as described in Koch, C. A. et al(1989) Mol. Cell. Biol. 9, 4131-4140.

The ligands for many receptor tyrosine kinase proteins are cell-bound,either as they are associated with the cell surface via heparin andhepatocyte growth factor or because they are transmembrane proteins(Lyman et al. 1993, supra). Accordingly, a ligand for Tek protein mayhave a cell-bound form. A cell-bound ligand may be identified byreacting the receptor tyrosine kinase protein of the invention, anisoform or a part thereof with a cell suspected of expressing the ligandon the surface of the cell following the procedures generally describedin Lyman et al., 1993, (Cell 75;1157-1167). Thus, the invention providesa method for identifying cells expressing a surface bound ligand of Tekprotein and for specifically selecting for such cells.

By way of example, a cDNA encoding a ligand for Tek protein may becloned by first constructing a fusion protein. The fusion protein mayconsist of the extracelluar domain of Tek protein (amino acids 19 to744, SEQ ID NO: 6 and FIG. 11B). The fusion protein may be expressed andused as a probe to examine cells or cell lines for their capacity tobind the extracelluar domain of Tek protein (determined by flowcytometry). The identification of cells and cell lines that bind theextracellular domain may be facilitated by incorporating in the fusionprotein a sequence encoding a marker protein for example, the Fc portionof human IgG which may be detected with labelled anti-human IgGantibodies. Cells or cell lines which bind the extracellular domain arepresumed to express a cell-bound form of the ligand.

Following identification of a source of the Tek ligand, a cDNAexpression library is constructed, following known techniques, usingmRNA from the cells/cell lines which have been identified as binding thefusion protein containing the extracellular domain of Tek protein. cDNAsare then transfected into host cells which are then screened for theircapacity to bind the extracellular domain of Tek protein. Individualclones which are capable of binding the extracellular domain of Tekprotein are identified and the cDNAs are sequenced. The cDNAs may beused as hybridization probes to isolate genomic DNA encoding the ligand.

The invention also provides a method of using the novel proteins of theinvention for assaying a medium for the presence of a substance thataffects a tek effector system. In particular the method may be used todetect a suspected agonist or antagonist of a tek effector system. Theagonist or antagonist may be an endogenous physiological substance or itmay be a natural or synthetic drug.

The term "tek effector system" used herein refers to the interactions ofa ligand, and the receptor tyrosine kinase protein of the invention, andincludes the binding of a ligand to the receptor protein or anymodifications to the receptor associated therewith, to form aligand/receptor complex and activating tyrosine kinase activity therebyaffecting signalling pathways, particularly those involved in theregulation of angiogenesis.

In accordance with one embodiment, a method is provided which comprisesproviding a known concentration of a receptor tyrosine kinase protein ofthe invention, isoforms thereof, or part of the protein, incubating theprotein, isoforms thereof, or part of the protein, with a ligand whichis capable of binding to the protein, isoforms thereof, or part of theprotein, and a suspected agonist or antagonist substance underconditions which permit the formation of ligand-receptor proteincomplexes, and assaying for ligand-receptor protein complexes, for freeligand or for non-complexed proteins.

The ligand-receptor complex, free ligand or non-complexed proteins maybe assayed as described above. Suitable ligands used in the assay methodmay be identified using the methods described above. The ligand may be anatural ligand or a synthetic derivative having similar biologicalactivity.

The invention also makes it possible to screen for antagonists thatinhibit the effects of an agonist of a tek effector system, but do nothave any biological activity in the tek effector system. Thus, theinvention may be used to assay for a substance that competes for thesame ligand-binding site on the novel receptor tyrosine kinase proteinof the invention.

It will be understood that the substances that can be assayed using themethods of the invention may act on one or more of the binding sites onthe receptor tyrosine kinase or the ligand, including agonist bindingsites, competitive antagonist binding sites, non-competitive antagonistbinding sites or allosteric sites.

The methods of the invention make it possible to screen a large numberof potential ligands for their ability to bind to the novel receptortyrosine kinase protein of the present invention. The methods of theinvention are therefore useful for identifying potential stimulators orinhibitors of angiogenesis, cardiogenesis or tumorigenesis.

The invention further contemplates a method for identifying a substancewhich is capable of binding to an activated receptor tyrosine kinaseprotein of the invention or an isoform or part of the activated protein,comprising reacting an activated receptor tyrosine kinase protein of theinvention, or an isoform, or part of the protein, with at least onesubstance which potentially can bind with the receptor tyrosine kinaseprotein, isoform or part of the protein, under conditions which permitthe formation of substance-receptor kinase protein complexes, andassaying for substance-receptor kinase protein complexes, for freesubstance, for non-complexed receptor kinase proteins, or forphosphorylation of the substance.

An activated receptor tyrosine kinase protein of the invention, orisoform or part thereof may be prepared by binding of a ligand to theextracellular domain of a receptor tyrosine kinase protein of theinvention which results in activation of the catalytic domain. Such aligand may be identified using the methods hereinbefore described. Anactivated receptor or part thereof, may also be prepared using themethods described for example in Reedijk et al. The EMBO Journal,11(4):1365, 1992 for producing a tyrosine phosphorylated receptor orpart thereof.

Conditions which permit the formation of substance-receptor proteincomplexes may be selected having regard to factors such as the natureand amounts of the substance and the receptor protein. Thesubstance-receptor complex, free substance or non-complexed proteins maybe isolated by conventional isolation techniques described above.Phosphorylation of the substance may be determined using for example,labelled phosphorous as described above.

In an embodiment of this method, intracellular ligands such as Srchomology region 2 (SH2)-containing proteins which are capable of bindingto a phosphorylated receptor tyrosine kinase protein of the inventionmay be identified. SH2-containing proteins refers to proteins containinga Src homology region 2 which is a noncatalytic domain of ˜100 aminoacids which was originally identified in the Vfps and Vsrc cytoplasmictyrosine kinases by virtue of its effects on both catalytic activity andsubstrate phosphorylation (T. Pawson, Oncogene 3, 491 (1988) and I.Sadowski et al., Mol. Cell. Biol. 6, 4396 (1986)). (See also Koch etal., Science 252:668, 1991; Moran et al., PNAS USA 87:8622 and Andersonet al., Science 250:979, 1990 for discussions on SH2-containing proteinsand the role of SH2 domains). SH2-containing proteins may functiondownstream of the Tek signalling pathway by binding to the activatedreceptor protein. Intracellular ligands which may be phosphorylated bythe novel receptor tyrosine kinase protein of the invention may also beidentified using the method of the invention.

The invention further provides a method for assaying for a substancethat affects angiogenesis, cardiogenesis, or tumorigenesis comprisingadministering to a non-human animal or to a tissue of an animal, asubstance suspected of affecting angiogenesis, cardiogenesis, ortumorigenesis and detecting, and optionally quantitating, the novelreceptor tyrosine kinase of the invention in the non-human animal ortissue.

In another embodiment, the method may be used to assay for a substancethat affects tumorigenesis, comprising administering a substancesuspected of affecting tumorigenesis to a non-human animal model oftumorigenesis and detecting, and optionally quantitating, the novelprotein kinase of the invention in the non-human animal. For example,the 3T3 cell transformation model in nude mice may be employed.

Substances which are capable of binding to the Tek protein of theinvention or parts thereof, particularly ligands, and agonists andantagonists of the binding of ligands and Tek protein, identified by themethods of the invention, may be used for stimulating or inhibitingangiogenesis or cardiogenesis, or inhibiting tumorigenesis. The efficacyof these substances in the treatment of human conditions may beconfirmed using non-human animal models, for example the models oftumorigenesis described above.

Cells, tissues, embryos, and non-human animals lacking in Tek expressionor partially lacking in Tek expression may be developed usingrecombinant molecules of the invention in particular recombinantmolecules containing sequences encoding the Tek protein having specificstructural mutations such as replacement, deletion or insertionmutations in the Tek gene, or having one or more regulatory elementswhich differ from the transcriptional and translation elements of thenative Tek protein. For example, the extracelluar domain or partsthereof, the transmembrane region or parts thereof; the tyrosine kinasedomain or parts thereof, and; the carboxy terminal tail may be deleted.A recombinant molecule may be used to inactivate or alter the endogenousgene by homologous recombination, and thereby create a Tek deficientcell, tissue or animal. The recombinant molecule may also contain areporter gene, as described herein, to facilitate monitoring ofexpression in the cells, tissues, etc.

Null alleles may be generated in cells, such as embryonic stem cells bya deletion mutation. A recombinant Tek gene may also be engineered tocontain an insertion mutation which inactivates Tek. Such a constructmay then be introduced into a cell, such as an embryonic stem cell, by atechnique such as transfection, electroporation, injection etc. Cellslacking an intact Tek gene may then be identified, for example bySouthern blotting, Northern Blotting or by assaying for expression ofTek protein using the methods described herein. Such cells may then befused to embryonic stem cells to generate transgenic non-human animalsdeficient in Tek. Germline transmission of the mutation may be achieved,for example, by aggregating the embryonic stem cells with early stageembryos, such as 8 cell embryos, in vitro; transferring the resultingblastocysts into recipient females and; generating germline transmissionof the resulting aggregation chimeras. Such a mutant animal may be usedto define specific nerve cell populations, developmental patterns ofcardiogenesis, and endothelial and highly vascularized tissue and invivo processes, normally dependent on Tek expression.

By way of example, specific targeted mutations may be employed togenerate a Tek receptor tyrosine kinase protein that is still competentto bind ligand, but which is unable to transduce a signal due to itslack of catalytic function. Such targeted mutations may be made in thehighly conserved intracellular cytoplasmic domain, for example, byaltering lysine⁸⁵³ to alanine⁸⁵³. A null allele of tek may also becreated by deletion of several nucleotides within an exon. For examplethe last 52 base pairs of exon-1 may be deleted.

The following non-limiting examples are illustrative of the presentinvention:

EXAMPLES

The following materials and methods were utilized in the investigationsoutlined in Examples I to VI:

DNAs

AKR/J, DBA, and AKR/J×DBA recombinant inbred mouse DNAs were obtainedfrom Jackson Labs (Bar Harbor, Maine), digested with AccI, blotted toZeta-Probe nylon membrane (Bio-Rad), and probed with the 1.6 kb tek cDNAlabelled by random priming (Feinberg, A. P. & Vogelstein, B. (1983)Analyt.Biochem., 132, 6-13). Hybridization was performed overnight at65° in 200 mM sodium phosphate pH7.0, 7% sodium dodecyl sulfate (SDS),1% bovine serum albumin (BSA), and 1 mM EDTA. Filters were washed twiceat 55° in 2×SSC (1×SSC=0.15 M NaCl, 0.015 M sodium citrate pH7.0) and0.1% SDS and twice in 0.2×SSC and 0.1% SDS, and exposed overnight toKodak XAR-5 film.

Mice

Embryos and adult mouse tissues were obtained from random bred CD-1stocks (Charles River, Quebec). Embryos were staged as Day 0.5 on themorning of a vaginal plug.

RNA Purification and Analysis

Total RNA was extracted from pools of 30 to 40 Day 9.5 and 12.5 murineembryonic hearts with RNAzol (CINNA/B10TECX Lab. Int.), with some addedmodifications. Briefly, tissues were washed with ice cold phosphatebuffered saline (PBS) and homogenized in 2.5 ml of RNAzol. Chloroform(250 μl) was added and the tubes were mixed vigorously and then chilledon ice for 15 min. The suspension was centrifuged for 15 min at 4° afterwhich the aqueous phase was collected and re-extracted twice more withphenol/chloroform/isoamyl alcohol (25:24:1; vol:vol:vol). The RNA wasprecipitated with an equal volume of isopropanol, collected bycentrifugation, and the pellet resuspended in diethylpyrocarbonate(DEPC)-treated 0.4 M sodium acetate, pH5.2. The RNA were thenreprecipitated with two volumes of 95% ethanol, washed with 70% and 95%ethanol, dried, and resuspended in DEPC treated 0.3 M sodium acetate,pH5.2. The RNA concentration was determined and the RNA stored at -70°until use.

Poly A--containing RNA was purified from a pool of 100 to 150 Day 12.5murine embryonic hearts with a QuickPrep mRNA isolation kit (Pharmacia)as outlined by the supplier.

For Northern blot hybridization, 5 μg of poly A--containing RNA from12.5 day embryonic heart was electrophoresed through aformaldehyde-agarose gel and blotted to a Zeta-Probe nylon membrane(Bio-Rad) according to established protocols (Sambrook et al., 1989,Molecular Cloning. Cold Spring Harbor Laboratory Press). The membranewas hybridized with a [³² P]-labelled antisense riboprobe synthesizedfrom the 1.6 kb tek cDNA in run off reactions with SP6 RNA polymerase(Promega).

Reverse Transcription Coupled to the Polymerase Chain Reaction (RT-PCR)

First strand cDNA was synthesized in a total reaction volume of 20 μlcontaining 20 μg of total RNA, 200 units of Mo-MLV-reverse transcriptase(BRL), either 1 μg of oligo-d(T)₁₈ (Day 12.5 RNA) (Boerhinger Mannheim)or 2 μg of random hexamer primers (Day 9.5 RNA) (Boerhinger Mannheim),1×PCR buffer (Cetus), 2.5 mM MgCl₂, 1 mM of dNTPs (Pharmacia), 40 unitsof RNAsin (Promega), and 12.5 mM dithiothreitol. The RNA was heated to65° C. for 10 min and cooled quickly on ice prior to addition to thereaction components. The reaction was allowed to proceed for 1 h at 37°and then terminated by heating for 5 min at 95°. For PCR, the reactionmixture was adjusted to a final volume of 100 μl containing 1×PCRbuffer, 1.5 mM MgCl₂, 800 μM dNTPs, and 1 μg of each of the twodegenerate tyrosine kinase oligonucleotide primers described by Wilks,A. F. (1989) Proc.Natl.Acad.Sci., 86, 1603-1607. Amplification wasperformed with a Ericomp thermocycler using the following parameters:denaturation for 2 min at 94°, annealing for 2 min at 42°, and extensionfor 4 min at 63°. After 40 cycles, the reaction products were collectedby ethanol precipitation and electrophoresed through at 2% low-meltagarose (Sea Plaque) gel. In most cases a band of approximately 200 bpwas visible within a background smear of ethidium bromide staining. Thisband was excised and recovered by three cycles of freeze-thaw in 100 μlof water. 10 μl of this solution was then subjected to a second round ofPCR under the same conditions described above.

Cloning and Sequencing of RT-PCR Products

After the second round of amplification, 10 μl of the reaction mixturewere analyzed on a gel for successful amplification. The remaining 90 μlwere then ethanol precipitated, digested with EcoRI and BamHI, gelpurified, and ligated to pGEM7Zf+ (Promega) digested with the sameenzymes. The ligation mixture was then transformed into MV1190 competentcells, individual ampe colonies picked, plasmid DNA prepared, and thecDNA inserts analyzed by single track dideoxynucleotide sequencing(Sanger, F., Nicklen, S. & Coulson, A. R. (1977). Proc.Natl.Acad.Sci.,74, 5463-5467). A single representative clone of each multiple isolatewas sequenced in its entirety. Of the 58 clones analyzed, roughly 10%showed no sequence identity to tyrosine kinases and were disregarded.

Isolation of Additional tek cDNA Sequences

Approximately 10⁶ plaques from an amplified, random primed 13.5 daymurine embryonic λgt10 cDNA library were hybridized with the 210 bp tekPCR product labelled with [³² P]-dCTP by PCR. Hybridization was carriedout overnight at 55° in 50% formamide, 10% dextran sulfate (Pharmacia),0.5% BLOTTO, 4×SSPE (1×SSPE=0.18 M NaCl, 10 mM NaH₂ PO₄, 1 mM EDTA,pH7.4), 100 μg/ml sheared salmon sperm DNA, and 2×10⁶ cpm/ml of probe.Filters were washed at 55° twice in 2×SSC containing 0.1% SDS and twicein 0.2×SSC containing 0.1% SDS, dried, and exposed overnight to KodakXAR-5 film. One clone was isolated from this screen and was found tocontain a 1.6 kb cDNA. The sequence of the 1.6 kb cDNA was determined bythe method of Sanger et al. (1977) from a set of anchored deletionsgenerated with a standardized kit (Erase-A-Base, Promega).

In Situ Hybridization

Embryos isolated on Day 12.5 were dissected away from all extraembryonictissues whereas embryos at earlier time points were recovered in utero.Embryos and adult tissues were fixed overnight in 4% paraformaldehyde,dehydrated with alcohols and xylenes, and embedded in paraffin. Tissueswere sectioned at 6 μm thickness and mounted on3-aminopropyltriethoxysilane treated slides (Sigma). After removal ofparaffin the samples were treated with predigested pronase (BoerhingerMannheim), acetylated with triethanolamine, dehydrated, and hybridizedaccording to the protocol described by Frohman, N. B., Boyle, M. &Martin, G. R. (1990), Development, 1101 589-607.

Dark and bright field photomicroscopy was performed with a Leitz VarioOrthomat 2 photomicroscopic system. Adjacent sections probed with a teksense probe produced no detectable signal above background.

Whole-mount in situ hybridizations were performed using a modificationof existing procedures (Tautz, D. & Pfeifle, C. (1989). Chromosoma,98,81-85; Hemmati-Brivanlou, A., Franck, D., Bolce, M. E., Brown, B. D.,Sive, H. L. & Harland, R. M. (1990). Development, 110, 325-330; Conlonand Rossant, in prep.). The hybridization of single-stranded RNA probeslabelled with digoxigenin was detected with antidigoxigenin antibodiescoupled to alkaline phosphatase. The En2 cDNA was prepared as set forthin Joyner A. L. & Martin, G. R. (1987). Genes and Dev., 1, 29-38 andexpression of En2 is described in Davis, C. A., Holmyard, D. P., Millen,K. J. & 2JJoyner, A. L. (1991) Development, 111:, 287-298.

lmmunohistochemistry

Sections were stained immunohistochemically for von Willebrand factorwith a commercially available kit (Biomeda). After color development,slides were counterstained with Harris hematoxylin.

EXAMPLE I

Isolation and Characterization of tek from a Day 13.5 Total Mouse EmbryocDNA Library

To identify and characterize tyrosine kinases expressed during murinecardiogenesis, cDNAs were synthesized from 9.5 and 12.5 day embryonicheart RNA by RT-PCR using degenerate oligonucleotide primers previouslydemonstrated to amplify tyrosine kinase sequences preferentially (Wilks,A. F. 1989, Proc.Natl.Acad.Sci., 86, 1603-1607). Considerable cellulardifferentiation and morphogenesis have occurred within the cardiacregion of the embryo by Day 9.5. At this stage the heart has developedfrom the primordial mesoderm cells of the cardiac plate into a primitivebent tube structure, consisting of two endothelial tubes enclosed withinthe developing myocardium. Between Day 9.5 and 12.5 the heart undergoesadditional complex morphological changes in association with theformation of the four chambers and septa characteristic of the adultheart. Sequence analysis of 58 clones obtained following amplificationrevealed that whereas roughly 10% did not contain sequence similaritiesto protein kinases the remainder corresponded to 5 distinct cDNAS (Table1--Identity and number of tyrosine kinase cDNA clones recovered from Day9.5 and 12.5 murine embryonic heart by RT-PCR). Four of these cDNAsrepresented previously characterized tyrosine kinases including, bmk,c-src, c-abl, and the platelet derived growth factor receptor β-subunit(pdgfrb). The isolation of bmk, c-src, and c-abl is consistent with thebroad tissue distribution of these kinases (Wang, J. Y. J. & Baltimore,D. (1983). Mol.Cell.Biol., 3, 773-779; Ben-Neriah et al., (1986). Cell,44, 577-586; Holtzman, D., Cook, W. & Dunn, A. (1987).Proc.Natl.Acad.Sci., 84, 8325-8329; Renshaw, M. W., Capozza, M. A. &Wang, J. Y. J. (1988). Mol.Cell.Biol., 8, 4547-4551). The recovery fromembryonic heart of pdgfrb at a relatively high frequency may indicatethat pdgfrb plays an important role in cardiogenesis, as has beensuggested by recent studies demonstrating that the addition of PDGF-BBto explants of axolotol cardiac field mesoderm stimulates the productionof beating bodies (Muslin, A. J. & Williams, L. T. (1991). Development,112, 1095-1101) the fifth cDNA, which was also isolated at highfrequency, was novel and for reasons that will become clear below wasdesignated tek. The 210 bp RT-PCR-derived tek clone was subsequentlyused to isolate additional tek cDNA sequences.

FIG. 2 (or SEQ ID NO:3) shows the nucleotide sequence of a 1.6 kb tekcDNA isolated from a 13.5 day mouse embryo cDNA library. Translation ofthis sequence reveals a single large open reading frame that terminateswith TAG at nucleotide 907, followed by 696 nucleotides of 3'untranslated sequence. Several features of the deduced amino acidsequence (or SEQ ID NO:4) suggest that the 1.6 kb tek cDNA encodes thecytoplasmic portion of a transmembrane RTK, consisting of the catalyticdomain followed by a short carboxy-terminal tail of 33 amino acidresidues.

FIG. 3 shows a comparison of the deduced amino acid sequence of tek (orSEQ ID NO:4) with that of other tyrosine kinases; Identical sequencesare denoted by periods. Dashes were added to allow for optimalalignment. The kinase insert and conserved regions of the catalyticdomain are indicated beneath the aligned sequences (Hanks, S. K., Quinn,A. M. & Hunter, T. (1988), Science, 241, 52). Comparative sequencesshown are for human Ret (SEQ ID NO:15) (Takahashi, M. & Cooper, G. M.(1987). Mol.Cell.Biol., 7, 1378-1385), and Jtk14 (SEQ ID NO:14)(Partanen, J., Makela, T. P., Alitalo, R., Lehvaslaiho, H. & Alitalo, K.(1990) Proc.Natl.Acad.Sci., 87, 8913-8917) and murine Flg (SEQ ID NO:16)(Reid, H. H., Wilks, A. F. & Bernard, O. (1990) Proc.Natl.Acad.Sci., 87,1596-1600).

As shown in FIG. 3, the putative kinase domain contains several sequencemotifs conserved among tyrosine kinases, including the tripeptide motifDFG, which is found in almost all known kinases, and the consensusATP-binding site motifs GXGXXG (SEQ ID NO:7) followed by AXK 16 aminoacid residues downstream (Hanks et al., 1988). Transmembrane RTK'spossess a methionine residue within the motif WMAIESL of conservedregion VIII of the catalytic domain (Hanks et al., 1988) as does tek,and the catalytic domain is interrupted by a putative 21 amino acidkinase insert, a structural motif not found in cytoplasmic tyrosinekinases (Hanks et al., 1988).

Comparison with other tyrosine kinases (FIG. 3) reveals that the deducedtek amino acid sequence shows 42% sequence identity to the mousefibroblast growth factor receptor Flg (Reid et al., 1990; Safran, A.,Avivi, A., Orr-Urtereger, A., Neufeld, G., Lonai, P., Givol, D. &Yarden, Y. (1990). Oncogene, 5, 635-643, Sambrook, J., Fritsch, E. F. &Maniatis, T. (1989). Molecular Cloning. Cold Spring Harbor LaboratoryPress) and 45% to the transmembrane RTK encoded by the human c-retproto-oncogene (Takahashi & Cooper, 1987). In addition, strikingsequence identity is observed to a 65 amino acid residue sequenceencoded by Jtk14, a putative tyrosine kinase cDNA isolated fromdifferentiating human K562 cells by RT-PCR (Partanen et al., 1990).Taken together, the results suggest that tek encodes a novel RTK.

EXAMPLE II

Chromosomal Mapping of the tek Murine Locus

Mapping of the tek locus in mice was accomplished by monitoring thestrain distribution pattern of an AccI restriction site polymorphism inrecombinant inbred (RI) mouse strains derived from matings between AKR/J(A) and DBA/2J (D) mice. The tek cDNA detects bands of 6.5, 6.1, 1.3 and6.5, 3.1, 1.3 kb in DNA from the A and D strains, respectively. Southernblot hybridization analysis of DNA from 24 RI mice with the 1.6 kb cDNAprobe, and comparison of the segregation pattern with the JacksonLaboratory data base, revealed 95.8% cosegregation between tek and bothbrown and pmv-23, two loci that have previously been localized to mousechromosome 4 (Lyon & Searle, 1989). Table 2 shows the cosegregation ofthe tek, brown, and pmv-23 loci in A×D strains. In Table 2 for each RIstrain, the symbol shown indicates the presence of an allelecharacteristic of the progenitor from which the strain was derived (A,AKR/J; D, DBA/2J). These data place tek between the brown and pmv-23loci within 3.8±1.9 centimorgans of each interval.

EXAMPLE III

Multiple tek-related Transcripts are Expressed in Embryonic Heart

Tek expression in embryonic heart was examined by Northern blothybridization using an antisense probe derived from the 1.6 kb tek cDNA.FIG. 4 shows a Northern blot hybridization analysis of tek expression in12.5 day murine embryonic heart; Arrows on the left denote the positionof migration of 28 S and 18 S ribosomal RNAs obtained from adjacent laneloaded with total RNA.

10 μg of yeast tRNA (lane 1) and 10 μg of total RNA from Py4-1 (lane 2),EOMA (lane 3) and MAE 22106 (lane 4) cells were hybridized in solutionwith [³² P]labelled tek, flk-1, and β-actin antisense RNA and digestedwith RNAse. Individual probes were added to RNA prepared from EH13.5(lanes 5 to 7). Digestion products were analyzed on a 6% sequencing geland autoradiographed for 24 hrs (lanes 5-7) and 48 hrs (lanes 1 to 4).The β-actin lanes were exposed for equal times. Relevant regions of thegel are shown.

FIG. 4 shows that the tek probe detects 4 transcripts of 4.5, 2.7, 2.2,and 0.8 kb in size in cardiac RNA from 12.5 day mouse embryos. Thesehybridizing species vary considerably in signal intensity, suggestingthat they may differ in relative abundance, with expression of the 2.7and 2.2 kb transcripts occurring at significantly higher levels than the4.5 and 0.8 kb RNAs. While the exact relationship among thesetranscripts is unclear, it is possible that they arise by differentialsplicing, since the 1.6 kb tek cDNA detects a single genomic locus inmouse DNA by Southern blot hybridization at the same stringency.

EXAMPLE IV

In Situ Localization of tek Expression During Mouse Embryogenesis

To determine which cell types express tek during development, RNA insitu hybridization analyses were performed on mouse embryos with anantisense riboprobe synthesized from the 1.6 kb tek cDNA.

FIG. 5 shows the in situ hybridization analysis of tek expression in the12.5 day embryo; A. Dark field illumination of a para-sagittal section.Bar: 600 μm. B. and C. Bright and dark field illumination respectively,of the heart region taken from a mid-sagittal section. Bar: 300 μm. IVand VI, fourth and sixth aortic arches; A, atrium; BA, basilar artery;CV, caudal vein; E, endocardium; L, liver; M, leptomeninges; Ma,mandible; My, myocardium; PC, pericardial cavity; RA, renal artery; SS,sino-auricular septum; SV, sinus venosus; V, ventricle.

FIG. 5A shows that in 12.5 day mouse embryos, expression of tek isreadily detected in the heart, the leptomeninges lining the brain andspinal cord, and the inner lining of major blood vessels, including thecaudal vein and basilar and renal arteries. In addition, thin bands ofhybridization are observed in the intersomite regions, corresponding totek expression in the intersegmental vessels. Close examination of theregion of the developing heart (FIG. 5B and 5C) reveals that tek isexpressed in the endocardium, as well as in cells lining the lumina ofthe atria, the IV and VI aortic arches, the sinus venosus, and thesino-auricular septum. In addition, tek expression is observed innumerous small blood vessels perforating the liver and mandible. Theseobservations, together with the overall pattern of hybridization seen inthe 12.5 day embryo, demonstrate that tek is expressed in theendothelial cells of the tunica interna, the innermost lining of theblood vessels; hence the designation tunica interna endothelial cellkinase, tek.

More detailed information on tek expression was obtained throughanalysis of sections from earlier developmental stages. Hybridization to6.5 and 7 day embryos revealed that while tek is expressed strongly inthe inner lining of the small blood vessels and capillaries of thematernal decidua, no expression is observed in either the embryo itselfor the ectoplacental cone. The absence of tek expression at these stagesis consistent with the fact that at 6.5 to 7 days the embryo containsonly a small amount of mesoderm from which endothelial cells are knownto be derived.

FIG. 6 shows the expression of tek precedes that of von Willebrandfactor in 8.5 day embryos; Adjacent transverse sections through an 8.5day embryo fixed in utero were either hybridized in situ with an [³⁵S]-labelled tek probe or stained immunohistochemically for vonWillebrand factor. A. Bright field illumination of tek expression, Bar:300 μm. B. Dark field illumination of section in A. C. Highmagnification of a blood island, slightly out of the field shown in A,depicting silver grains over flat, elongated cells of endothelial-likemorphology, Bar: 50 μm. D. Adjacent section to A at higher magnificationshowing absence of expression of von Willebrand factor in the embryo,Bar: 100 μm. E. Adjacent section to A at higher magnification showingexpression of von Willebrand factor in the endothelial lining of theblood vessels of the maternal decidua. Bar: 200 μm. F. Highmagnification of cephalic region in A showing silver grains over alarge, round cell of angioblast-like morphology (arrow). Bar: 50 μm. G.Bright field illumination of a sagittal section of an 8.5 day embryohybridized in situ with an [³⁵ S]-labelled tek probe. Bar: 300 μm. H.Dark field illumination of G. I. Higher magnification of heart region inA showing silver grains over cells with endothelial- and angioblast-likemorphology in the developing endocardium. Bar: 100 μm. J. Highermagnification of somite region in A showing tek-expressing cellsextending beneath, and possibly from, the ventral surface of thesomites. Bar: 100 μm. A, amnion; Ag, presumptive angioblast; BI, bloodisland; D, maternal decidua; DA, dorsal aorta; E, endocardium; Ec,ectoplacental cone; En, endothelial cell; G. foregut; HV, head vein; NF,neural fold; S, somite; Y, yolk sac.

RNA in situ analysis of 8.0 day embryos revealed that tek expressionfirst becomes detectable in the developing yolk sac and a few smallclusters of cells in the cephalic mesenchyme. This expression becomesmore pronounced by Day 8.5, at which time significant hybridization canbe observed in the mesodermal component of the amnion (outer cell layer)and yolk sac (inner cell layer), as well as in the developingendocardium and the inner lining of the head veins and dorsal aortae(FIG. 6A and 6B). In addition, sagittal sections reveal numerous focalareas of hybridization throughout the cephalic mesenchyme in regionsthought to contain developing vasculature, as well as a small number oftek-expressing cells extending beneath the ventral surface of thesomites (FIG. 6H and 6J).

Whole mount in situ hybridization analysis confirmed and extended theabove observations, as well as provided a three dimensional perspectiveon tek expression during embryogenesis. FIG. 7 shows tek expression inwhole mount embryos; A., B., C. and D. tek expression in Day 8.0embryos. E. tek mRNA distribution in a Day 9.5 embryo. F. En2 expressionin a Day 8 embryo. I, II, III, first, second and third aortic arches;DA. dorsal aorta; E, endocardium; G, foregut pocket; H, heart; IS,intersegmental vessel; My, myocardium; ; NF, neural fold; OT; oticvesicle; V, vitelline vein; Y. yolk sac. Bars: 250 μm.

Consistent with our observations with sectioned material, localized tekexpression was not observed on embryonic Day 7. The first detectableexpression was seen about the time of first somite formation when signalwas observed in the yolk sac, head mesenchyme, and heart. In Day 8.5embryos, tek was found to be expressed in these same areas, and in thepaired dorsal aortae, the vitelline veins, and in the formingintersegmental vessels (FIG. 7). By this time, tek expression wasclearly confined to blood vessels within the embryo. On Day 9, tekexpression was seen in addition, in the aortic arches and expression wasvery striking in the endocardium (FIG. 7E). Control hybridizations withan En-2 probe demonstrated the specificity of tek RNA detection (FIG.7F).

EXAMPLE V

Expression of tek in Endothelial Cell Progenitors

The observation that tek is expressed between Day 8.0 and 8.5 in focalregions thought to represent developing blood vessels raised thepossibility that tek might be expressed in endothelial cell progenitors.Indeed, close inspection of hybridized sections from 8 to 8.5 dayembryos revealed that while the expression of tek in the maternaldecidua is restricted to cells of an endothelial cell morphology, tekexpressing cells in the embryo are of two morphologically distinct celltypes. In the developing blood islands of the yolk sac, where tekexpression is first detected, silver grains are localized predominantlyto elongated cells with characteristic endothelial cell morphology (FIG.6C). In contrast, within the cephalic mesenchyme, silver grains arefrequently observed over large, round cells that, on the basis ofsimilar morphology to cells described during avian embryogenesis(Pardanaud et al., 1987; Coffin & Poole, 1988; Noden, 1989; Noden,1991), correspond to angioblasts, the presumptive progenitor ofendothelial cells (FIG. 6F). Both cell types are observed in thedeveloping endocardium (FIG. 6I) which, at later stages, is known tocontain only fully mature endothelial cells.

To characterize more precisely the staging of tek expression within theendothelial lineage, sections adjacent to those used for in situhybridization were stained immunohistochemically for von Willebrandfactor, a well characterized marker of mature endothelial cells (Jaffe,E. A., Hoyer, L. W. & Nachman, R. L. (1973). J.Clin.Invest., 52,2757-2764; Hormia, M., Lehto, V.-P. & Virtanen, I. (1984),Eur.J.Cell.Biol., 33, 217-228). FIG. 6B and H shows that whereas tek isexpressed in both the maternal decidua and the embryo at Day 8.5,expression of von Willebrand factor is observed only in thetek-expressing, vascular endothelial cells of the maternal decidua (FIG.6D and 6E). Hence tek expression precedes that of von Willebrand factorduring embryogenesis. The same scenario is observed at laterdevelopmental stages during vascularization of individual organs.

FIG. 8 shows the expression of tek precedes that of von Willebrandfactor in the developing leptomeninges; A. Absence ofimmunohistochemical staining of von Willebrand factor in Day 12.5leptomeninges. Arrow denotes a large blood vessel faintly positive forvon Willebrand factor. B. In situ detection of tek expression in Day12.5 leptomeninges. C. Staining of von Willebrand factor in Day 14.5leptomeninges. Day 14.5 leptomeninges were positive for tek expression(not shown). M, leptomeninges. Bars: 200 μm.

FIG. 8 shows that in the 12.5 day embryo, the developing leptomeningeshybridizes strongly with tek but fails to stain positive for vonWillebrand factor. By Day 14.5, however, expression of von Willebrandfactor can be readily detected in the leptomeninges. Assuming that thereis not a significant lag between transcription and translation of vonWillebrand factor, these observations, together with those on themorphology of tek-expressing cells, suggest that tek is expressed inboth mature endothelial cells and their progenitors.

EXAMPLE VI

tek is Expressed in Adult Vasculature

While the above results establish that tek is expressed duringvascularization of the embryo, it was also of interest to determinewhether expression of tek is maintained in endothelial cells of theadult. In situ hybridization analysis of a section through the heartregion of a 3 week-old mouse revealed that tek is expressed in theendocardium as well as in the endothelial lining of major blood vessels,both arteries and veins, connecting with the adult heart (FIG. 9).

FIG. 9 shows the expression of tek in adult vasculature. A. Bright fieldillumination of a section through the upper heart region of a 3 week-oldmouse hybridized with an [³⁵ S]-labelled tek probe. Bar: 20 μm. B. andC.

Bright field illumination showing tek expression in endothelial cellslining the artery and vein respectively. Bar: 1 μm. Immunohistochemicalstaining of adjacent sections revealed that structures positive for tekexpression also stained positive for von Willebrand factor. A, artery;Bl, extravasated blood; T, trachea; V, vein.).

The intensity of the hybridization signal observed for these structuresis considerably lower than that observed for the endocardium and bloodvessels of 12.5 day embryos hybridized and processed in parallel. Thiscould indicate that mature endothelial cells, which are thought to beresting, have a different quantitative or qualitative requirement forexpression of tek.

EXAMPLES VII to X

The following materials and methods were utilized in the investigationsoutlined in Examples VII to X:

DNAs

Tek- and tie-specific probes corresponding to sequences encoding theFNIII repeats (see FIG. 13A) were prepared as follows: The tek cDNA wasdigested with Pst I to yield a 0.95 kb fragment spanning sequences N1399to 2344 (see FIG. 11B and SEQ ID NO:5). The tie-specific probe wasgenerated by reverse transcription linked to PCR with two tie-specificoligonucleotides designed from the published sequence (5'¹²⁸⁸TTGCGGACAGTGGGTTCTGGGAGT (SEQ ID NO:9) and 5'²⁴¹⁴CGATGCAGGCAGCTTCTGCGGAT (SEQ ID NO:10)) and RNA prepared from the humanleukemia cell line, KG-1, which was previously shown to express tie(Partanen et al., Mol. Cell Biol. 12:1698-1707, 1992). First strandsynthesis was done with random Hexamers (Pharmacia) according toestablished protocols (Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989). The cDNA/RNA mixture was then treated with 0.1 NaOH,neutralized with HCl, and an aliquot used as template for PCR. PCR wasperformed in 100 μl containing: 10 μl of 10× reaction buffer 1(Stratagene), 10 μl dimethyl sulfoxide, 2 μg of each oligonucleotide,200 μM of dNTPs and 2.5 units of Pfu polymerase (Stratagene). Thisreaction mixture was then cycled 30 times as follows: 94° for 45 sec,52° for 2 min, 72° for 3 min, after which the products were resolved ina low-melt agarose gel. A major species of 1.1 kb was excised andrestriction mapped to ascertain that the correct DNA fragment had beenamplified. The tie-specific cDNA was amplified by PCR and purified asabove.

Probes were labelled by random priming (Feinberg et al., Anal. Biochem.132:6-13, 1983) with a kit according to the protocol supplied by themanufacturer (Pharmacia). Hybridization to immobilized DNA was performedovernight at 65° in 200 mM sodium phosphate pH7.0, 7% sodium dodecylsulphate (SDS; BDH), 15% Formamide (BDH), 1% bovine serum albumin (BSA;Sigma), and 1 mM EDTA. Filters were washed twice at 55° in 2×SSC(1×SSC=0.15 M NaCl, 0.015 M sodium citrate pH7.0) and 0.1% SDS and twicein 0.2×SSC and 0.1% SDS, and exposed overnight to Kodak XAR-5 film.

Mice

Embryos and adult mouse tissues were obtained as described above forExamples I to VI.

RNA Purification and Analysis

Total RNA was extracted from cell pellets with RNAzol (CINNA/BIOTECXLab. Int.) as described above for Examples I to VI. Poly A--containingRNA was purified from a pool of 150 Day 12.5 murine embryonic heartswith a QuickPrep mRNA isolation kit (Pharmacia) as outlined by thesupplier.

Tek, flk-1, and β-actin transcripts were detected by RNAse protectionanalysis with a kit (Ambion) according to conditions recommended by thevendor. RNA antisense probes were generated by run-off transcriptionwith a kit (Promega) in the presence of ³² [P]-CTP (3000 Ci/mmol;Dupont)following subcloning of cDNA fragments into either pGEM7zf+ orpBluescript II SK--. Probes corresponded to sequences 2416 to 2683 forflk-1 (Matthews et al. Proc. Natl. Acad. Sci. USA 87:8913-8917, 1991),1257 to 1633 for tek and 883 to 970 for β-actin. The flk-1 sequenceswere isolated from a Day 13.5 embryo cDNA library. The β-actin probe wasprovided by F. Shalaby. Digestion products were resolved in a 6%sequencing gel containing 8 M urea.

cDNA Cloning

Poly A-selected RNA (5 μg) from Day 12.5 embryonic heart (EH12.5) wasused as template to make double-stranded cDNA using a You-Prime cDNASynthesis Kit (Pharmacia) as outlined by the supplier. The reversetranscription reaction was supplemented with 1000 units of Super ScriptMMLV reverse transcriptase (BRL). Double-stranded cDNAs were ligated toadaptors, fractionated in a low-melt agarose gel, and molecules 2 to 4.5kb in size were liberated by digestion with β-Agarase I (BioLabs). ThecDNAs were then precipitated with ethanol and ligated to EcoR I-digestedand dephosphorylated lambda Zap II arms (Stratagene). The ligationproducts were packaged in vitro using Gigapack II packaging extractsaccording to the protocol provided (Stratagene).

Filters containing the unamplified EH12.5 library (1.3×10⁶ plaques) werehybridized with clone 18a1 (see FIG. 11A). This screen produced 72positive clones, the two largest of which (FIG. 11A; clones 8C and 24B)were sequenced in their entirety. To clone the 5' end of tek, a nestedPCR strategy was employed on EH12.5 phage DNA using two tek-specificprimers designed to hybridize to sequences 887 and 912 (EC1 primer) and786 to 807 (EC2 primer) of the coding strand and a primer specific forthe T7 polymerase binding site within the phage arm. Template phage DNAfrom 10¹⁰ plaque forming units was purified by the polyethyleneagglutination procedure (Sambrook et al., 1989). The PCR reaction wasrun through a first cycle in the absence of T7 primer in a volume of 100μl containing 50 ng of phage DNA, and EC1 primer (1 μM) in 1×PCR buffercontaining 50 mM KCl, 10 mM Tris-Cl pH 8.3, 1.5 mM MgCl₂, 0.1% gelatin,200 mM dNTPs (Pharmacia). The DNA was denatured at 94° for 1 minute,annealed to the ECI primer at 55° for 1 minute, and reacted with Taqpolymerase (2.5 units; Cetus) for 2 minutes at 72°. The T7 primer wasthen added at 1 μM and PCR continued for 40 cycles under the sameconditions. The products were collected by ethanol precipitation andanalyzed on a 1.5% low-melt agarose gel (Seaplaque, FMC). A band ofapproximately 600 bp was visible within a background smear extending upto 2 kb. The 600 bp band was excised, released from the gel by β-agaraseI treatment as described by the supplier, digested with EcoR I (foundwithin the λZapII multiple cloning site) and Hind III found at position881 in the tek cDNA, and ligated to pGem7Zf+ (Promega) resulting inclone EC1A. The remainder of the PCR products from 0.6 to 2 kb wererecovered as above, and submitted to a second round of PCR using EC2 andT7 primers under the same conditions as described earlier. The longestproduct obtained, 800 bp, was subcloned in pGem4Z (Promega) afterdigestion with EcoR I and Sph I found within the overlapping 600 bp EC1Aclone resulting in clone EC2D.

To identify potential PCR-generated sequence artifacts, duplicatefilters containing the EH12.5 library were probed with the PCR generatedECLA clone and a 5' fragment of clone 24B (see FIG. 11A). Two cloneswere obtained which hybridized with both of these probes as well as theEC2 primer. These two clones, 11b and 13a (see FIG. 11A), were sequencedin their entirety.

The tek cDNA was sequenced on both strands using a T7 DNA-Pol sequencingkit (Pharmacia) according to conditions recommended by the vendor. Thecomplete sequence was deduced by sequencing subcloned cDNA fragments andby using tek-specific primers. The cDNA sequence has been deposited. inGenbank/EMBL under accession number X67553.

Tek Antibodies

A DNA fragment encoding the C-terminal 43 amino acid residues of Tek wasprepared by PCR and subcloned into pGEX3X (Pharmacia). Theglutathione-S-transferase-Tek (GST-Tek) fusion protein produced in E.coli was purified by affinity chromatography with glutathione-sepharose4B (Pharmacia) and used to immunize rabbits according to establishedprotocols (Harlow et al., Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press; Cold Spring Harbor, N.Y., 1988). Tek-specificserum was subsequently purified by absorption to a DHFR-Tek fusionprotein (Qiagen), containing the Tek cytoplasmic domain (nucleotides2254-3477), which had been cross-linked to CNBr activated Sepharose 4B.

Detection of the Tek Protein

COS cells were transfected by the calcium phosphate co-precipitationmethod (Chen et al., Mol. Cell Biol. 7:2745-2752, 1987) with anexpression plasmid, pcDtek, containing the tek cDNA cloned into the EcoRI site of pcDNA1 (Invitrogen). Transfected cells were allowed to recoverfor 16 hr in normal medium (DMEM containing 10% bovine calf serum),after which they were washed three times with PBS, and metabolicallylabelled for 16 hr with 200 μCi [³⁵ S]-methionine (DuPont, 1000 Ci/mmol)in 3 ml of methionine-deficient medium for 16 hours. The [³⁵ S]-labelledcells were washed 3 times with ice-cold PBS and lysed in RIPA buffer (50mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton-X100, 0.1% SDS, 1 mM EDTA)supplemented with 1 mM PMSF, 10 μg/ml leupeptin, 10 μg/ml pepstatin A,10 μg/ml aprotinin, and 10 μg/ml soya bean trypsin inhibitor. The cellslysates were cleared by centrifugation (12,000×g at 4° for 15 min) andthen incubated overnight at 4° with 4 μg of affinity purified Tekantibodies. The immunocomplexes were collected on protein A-sepharosebeads in RIPA buffer (Pharmacia), and then washed 3 times with RIPAbuffer, twice with LiCl wash buffer (50 mM Tris-Cl, pH 7.5, 200 mMLiCl), and once with RIPA buffer. The resulting immunocomplexes wereboiled in sample buffer and analyzed by SDS-PAGE. Radioactive proteinswere detected by fluorography (EN³ HANCE;DuPont).

For detection of Tek by Western blotting, lysates were prepared fromPy4-1 cells and Day 13.5 mouse embryonic hearts and umbilical veins byboiling of tissues and cell pellets in sample buffer, followed bysonication and centrifugation to remove insoluble material. The extractswere separated on a 7% SDS polyacrylamide gel, transferred tonitrocellulose filters and blocked overnight with 5% BSA and 1%ovalbumin in TBST (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.1% Tween 20).The blocked filters were then incubated with affinity purified Tekantibodies in the above blocking solution plus 0.5% SDS and 0.1% NP-40,washed extensively with TBST containing 0.5% SDS, reblocked with BSA andovalbumin in TBS, and finally incubated with Protein A-horseradishperoxidase conjugate (BioRad) in TBSN (50 mM Tris-Cl, pH7.5, 150 mMNaCl, 0.2% NP-40) at 1:500 dilution for 30 minutes. The filters werewashed 5 times in TBSN and developed by the ECL chemiluminescence method(Amersham).

Cells

The Py4-1, MAE 22106, and EOMA cell lines have been described previously(Dubois et al., Exp. Cell. Res. 196:302-313, 1991; Obeso et al., Lab.Invest. 63:259-269, 1990). Cells were cultured in DMEM containing 10%fetal bovine serum (Hy-Clone).

EXAMPLE VII

Isolation and Characterization of Tek from a Day 12.5 Embryonic MouseHeart Tissue cDNA Library

To acquire additional tek sequences, a cDNA library was constructed fromRNA prepared from Day 12.5 embryonic heart tissue. From 12tek-hybridizing clones that were subsequently selected andcharacterized, 7 different overlapping cDNAs were identified and used toassemble a contiguous tek cDNA of 4177 nucleotides (N) (FIGS. 11A and11B).

FIG. 11A is a schematic representation of the cDNAs used to assemble thetek cDNA and the predicted structure of the encoded gene product. Theregions corresponding to the Ig-like, EGF-like, and FNIII repeats aredepicted by hatched, stippled, and cross-hatched boxes, respectively.The transmembrane and kinase regions are depicted by solid and openboxes, respectively. SEQ ID NO: 5 and FIG. 11B shows the nucleotidesequence and deduced amino acid sequence of the 4177N tek cDNA. The twocysteines in each Ig-like loop are circled and the EGF-like repeats arebracketed. The beginning and end of each FNIII repeat are indicated byarrowheads. Both the putative signal peptide and transmembrane regionsare underlined; the kinase domain is framed by square brackets.

The assembly of this cDNA revealed that two of the overlappingtek-hybridizing clones, 13A and 8C, also contained sequences of unknownorigin. The novel sequences in the 3'-half of clone 8C contained stopcodons in all three reading frames, were extremely AT-rich, and bore norelationship to those of other tyrosine kinases. The novel 265N at the5' end of clone 13A were not represented in any other overlapping tekcDNA or genomic clones isolated; moreover, the point at which thesesequences diverged from those in genomic DNA did not correspond to aconsensus splice acceptor site. Therefore, while the possibility thatthe novel sequence in clones 13A and 8C are derived from the tek locuscould not be excluded, the simplest interpretation is that thesesequences were acquired as a cDNA cloning artifact.

Conceptual translation of the 4177N tek cDNA (SEQ ID NO: 5 and FIG. 11B)revealed a single large open reading frame extending from a putativeinitiation codon, ATG, at N124 to an in-frame stop codon, TAG, at N3490.The sequence surrounding the putative initiation codon conforms to theoptimum consensus sequence for initiation of translation (Kozak, NucleicAcids Res. 12:1451-1459, 1984). In addition, the 18 amino acid residuesencoded immediately after the putative initiation codon are sufficientlyhydrophobic to constitute a signal peptide. While the sequencesdownstream of the termination codon do not contain a polyadenylationsignal, they are fairly AT-rich, as is frequently characteristic of 3'untranslated sequences. Since stop codons are found in all three readingframes both upstream and downstream of the single large open readingframe, it follows that the 4177N tek cDNA probably contains all of theTek coding sequences.

FIG. 11A shows that the predicted 1,122 residue protein encoded by thetek cDNA has several structural motifs that, together, set it apart fromother RTKs. Within the extracellular domain, three distinct types ofstructural motifs can be identified, including immunoglobulin-likeloops, EGF-like repeats, and fibronectin type III (FNIII) repeats.Briefly, two immunoglobulin-like loops, with characteristically placedcysteines (Williams & Barclay, Annu. Rev. Immunol. 6:381-405, 1988), arepresent between residues 19 and 209 and 344 and 467 (SEQ ID NO: 5 andFIG. 11B). These two immunoglobulin-like loops are separated from oneanother by three tandem EGF-like repeats that show homology to similarmotifs found in other cell-surface proteins, such as Tie and Notch (FIG.12A). Moreover, the second immunoglobulin-like loop is followed by threeregions showing homology to FNIII repeats found in polypeptides such asDLAR and fibronectin (FIG. 12B).

FIGS. 12A and 12B shows a sequence comparison between Tek and Tie. FIG.12A is a sequence comparison of the Tek (SEQ ID NO:23) EGF-like repeats(SEQ ID NO:17-19) with those of Tie (SEQ ID NO:20-22) (Partanen et al.,1992), EGF (Gray et al., Nature, 303, 722-725, 1983) and Notch (SEQ IDNO:24) (Rebay et al., Cell, 67, 687-699, 1991). The consensus sequenceis written below. Upper case letters denote 100% conservation; lowercase letters, greater than 70% conservation. Conserved cysteines andglycines are denoted by open and cross-hatched boxes, respectively. FIG.12B is a sequence alignment of the three mouse Tek (SEQ ID NO:25, 27 and29) and human Tie (SEQ ID NO:26, 28, and 30) (Partanen et al., 1992)FNIII repeats with a representative FNIII repeat from Drosophila DLAR(SEQ ID NO:32) (Streuli et al., 1988; Streuli et al., 1989) and ratfibronectin (SEQ ID NO:34) (Scwarzbauer et al., 1987). The deducedconsensus is given below. Upper case letters denote 100% conservation;lower case letters, greater than 60% conservation.

The extracellular domain of Tek receptor tyrosine kinase protein is,therefore, particularly complex, representing a composite of threedifferent structural motifs that are usually not found collectivelywithin a single RTK.

Anchoring of Tek receptor tyrosine kinase protein in the membrane ismost probably achieved by the highly hydrophobic stretch of residuesthat extends between positions 745 and 771 and which is followed by thetwo basic residues, lysine and arginine (SEQ ID NO: 5 and FIG. 11B).When this putative transmembrane region is used to define the boundaryof the Tek extracellular domain, 8 consensus sites for potentialN-linked glycosylation are present within the extracellular portion ofthe molecule.

The catalytic region of Tek receptor tyrosine kinase protein, whichstarts at residue 829, is interrupted by a 21-amino acid insert atresidue 913 (SEQ ID NO: 5 and FIG. 11B). Interestingly, the kinaseinsert does not contain a tyrosine residue whose phosphorylation inother RTKs has been implicated as the site for binding of downstreamsubstrates (Anderson et al., 1990; Escobedo et al., 1991; Klippel etal., 1992). However, Tek does contain a 32-amino acid residue carboxyltail that contains tyrosine residues (FIG. 11B). Tek receptor tyrosinekinase protein may therefore mediate signal transduction by binding ofdownstream signalling molecules to these tyrosine residues when they arephosphorylated, as has been found for other RTKs, such as FGFR-1 andEGFR (Mohammadi et al., Mol. Cell. Biol. 11, 5068-5078, 1991; Margoliset al. EMBO. J., 9, 4375-4380, 19.

EXAMPLE VIII

Tek Expression in Cultured Endothelial Cells

The finding that tek expression is restricted to angioblasts andendothelial cells, both in the embryo and in the adult, promptedanalysis of several cell lines of endothelial origin for expression oftek. To obtain additional insight into the character of these cellpopulations, they were also examined for expression of flk-1 (Matthewset al. 1991), which encodes an RTK whose expression may also berestricted to cells of the endothelial lineage, but which appears toprecede that of tek by approximately one day in the developing embryo.

FIG. 14 shows the profile of tek and flk-1 expression detected by RNAseprotection analysis in Py4-1, a transformed cell line established from ahaemangioma originating in a polyoma middle T antigen-expressingtransgenic mouse (Dubois et al., 1991); EOMA, a cell line derived from aspontaneously arising haemangioma (Obeso et al., 1990); and MAE 22106,an endothelial cell line cultured from normal mouse aorta (Pendl et al.,Dev. Biol.). The results show that whereas both tek and flk-1 areexpressed in Day 13.5 embryonic heart, and in Py4-1 cells at relativelyhigh levels, the EOMA cell line expresses detectable levels of flk-1 butnot tek RNA, while the MAE 22106 cell line expresses detectable levelsof tek but not flk-1. The detection of tek and flk-1 transcripts inthese cell populations is consistent with the in situ hybridizationstudies showing that these two genes are expressed in cells of theendothelial lineage. However, the finding that both tek and flk-1 areexpressed at significant levels in only one of the three endothelialcell lines examined is of interest. This apparent discordance in tek andflk-1 expression in cell lines could reflect the intrinsic heterogeneitythat has been documented for endothelial cell populations cultured fromdifferent anatomical sites (Gerritsen, Biochem. Pharmacol., 36,2701-2711, 1987 Gumkowski et al., Blood Vessels, 24, 11-23, 1987), thedifferential retention of expression of these markers followingmalignant transformation or in vitro culture, or the differentialexpression of these two RTKs in different cell lines that correspond tocells of the endothelial lineage at different stages of differentiation.This latter possibility stems from the observation that expression offlk-1 not only precedes that of tek during embryogenesis but, also, thatflk-1 appears to be down-regulated in endothelial cells as theydifferentiate, whereas tek is not. In any event, these results providefurther evidence that tek and flk-1 are differentially regulated.

EXAMPLE IX

Expression of Tek Receptor Tyrosine Kinase Protein

To characterize the protein encoded by the tek cDNA, COS cells weretransfected with a mammalian expression vector containing tek (asdescribed above). Cell extracts prepared from metabolically labelledtransfectants were analyzed for Tek receptor tyrosine kinase proteinexpression by immunoprecipitation with affinity-purified antibodydirected against the carboxy terminal 43-amino acid residues.

FIGS. 15A and 15B shows that tek directs the synthesis of a 140 kDaprotein. FIG. 15A shows immunoprecipitation of Tek from transfected COScells. Untransfected (lane 1) and transfected (lane 2) COS cells werelabelled with [³⁵ S]-methionine and lysates were prepared. The lysateswere then subjected to immunoprecipitation with anti-Tek serum asdescribed above. Antibody specificity was determined by the addition of100 μg of GST-Tek fusion protein (competitor) to the antibody prior tothe addition of cell extract (lane 3). FIG. 15B is a Western analysis ofTek expression in COS, Py4-1 cells and in embryonic tissues. Proteinsamples from untransfected and transfected COS cells, umbilical vein,Py4-1 cells, and Day 13.5 embryonic heart tissue (lanes 1 to 5,respectively) were analyzed for the presence of Tek receptor tyrosinekinase protein using affinity purified Tek antibodies.

FIG. 15A shows that a 140 kDa protein was specifically precipitated fromtransfected but not untransfected COS cells. Moreover, this 140 kDaprotein could be detected immunologically by Western analysis (FIG. 15B,lane 2) and its immunoprecipitation could be competed by a GST fusionprotein containing the 43-residue carboxy terminal segment to which theantibody was raised (FIG. 15A, lane 3). The apparent size of the encodedTek protein, 140 kDa, is approximately 20 kDa greater than thatpredicted by the deduced amino acid sequence (126 kDa). The larger sizeof the detected protein presumably indicates that Tek is a glycosylatedcell surface protein.

The protein encoded by the tek cDNA in transfected COS cells wascompared with that encoded by the native gene in tissues and a cell linepreviously shown to express tek. FIG. 15B shows that cell lysatesprepared from umbilical vein, Py4-1 cells, and Day 13.5 embryonic heartall contained a 140 kDa protein that reacted specifically with Tekantibody and which comigrated with the species detected in transfectedCOS cells. A slightly faster migrating species was also detected inPy4-1 cells. This species most likely represents an incompletelyglycosylated form of Tek receptor tyrosine kinase protein, although itmay be a distinct cross-reacting polypeptide. Taken together, theseresults indicate that the tek cDNA shown in SEQ ID NO:5 and FIG. 11Bcontains the complete coding information for the native Tek receptortyrosine kinase protein.

EXAMPLE X

Tek is not the Murine Homolog of Tie

Tek shows some similarities to a human RTK, designated Tie, (Partanen etal., 1992). First, expression of tie was reported to be restricted toendothelial cells, as was observed for tek. Second, tie was mapped tohuman chromosome 1p33 to 1p34, a region which shows synteny with theinterval to which tek was mapped on mouse chromosome 4 (See Example II).And third, tie, unlike all previously described members of the RTKfamily, encoded a molecule with virtually the same multidomain structureas Tek receptor tyrosine kinase protein. In fact, comparison of theprimary structure of Tek and Tie proteins revealed considerable sequencesimilarity in the cytoplasmic region and the EGF-like repeats of theextracellular domain; however, this sequence similarity dropped offmarkedly in the immunoglobulin-like loops and the FNIII repeats (seeFIGS. 12A and 12B). The relatively low sequence similarity within thesesubregions implied that tek might not be the murine homolog of tie. Toresolve this issue, the pattern of hybridizing bands detected in digestsof mouse and human genomic DNA by tek and tie probes containingsequences corresponding to equivalent regions (the FNIII repeats) oftheir respective genes (see FIG. 13A) was compared.

FIGS. 13A and 13B show the relationship between tek and tie. FIG. 13Ashows the structural relationship between Tek and Tie. Structural motifsare depicted as described in respect to FIG. 11A and the numbers denoteper cent sequence similarity between corresponding regions of the tworeceptors. The bar indicates the cDNA region of tek and tie used asprobes in panel B. FIG. 13B shows a Southern analysis of mouse (lanes1,3,5, and 7) and human (lanes 2,4,6, and 8) DNAs digested with eitherPst I (lanes 1 to 4) or with BstX I (lanes 5 to 8). Immobilized DNAswere hybridized with either the tek- (lanes 1,2,5, and 6) or tie- (lanes3,4,7, and 8) specific probes depicted in FIG. 13A. The position of themolecular weight markers, (8.4, 7.2, 6.4, 5.7, 4.8, 4.3, 3.7, 2.3 and1.9 kb) are depicted to the left of the panel.

FIG. 13B shows that whereas the tek probe hybridized with 4 Pst Ifragments of 15.4, 10, 4.7, and 2.4 kb (lane 1) and 5 BstX I fragmentsof 12.2, 11.5, 9.6, 7, and 5 kb (lane 5) in digests of mouse DNA, thetie probe detected 2 PstI fragments of 5 and 2.1 kb (lane 3) and 2 BstXI fragments of 6.9 and 4.7 kb (lane 7). Consistent with these results,the tek and tie probes also hybridized with different sized Pst I andBstX I fragments in human DNA. Thus, tek and tie are distinct, butclosely related, members of a novel RTK gene subfamily.

EXAMPLE XI

Chromosomal Mapping of the Human Tek Locus

In situ hybridization was used to map the human tek gene. An XbaI-digestof ptek cDNA was labelled to a specific activity of 9×10⁷ cpm/μg DNAwith [³ H]-dTTP and [³ H]-dATP (New Englan Nuclear) using a multiprimeDNA labelling system (Amersham, #RPN1600Y). In situ hybridization toBrdU-synchronized peripheral blood lymphocytes was performed using themethod of Harper and Sanders (1981, Chromosoma 83:431). Briefly,metaphase chromosomes on slides were denatured for 2 minutes at 70° C.in 70% deionized formamide, 2× SSC. Slides were then dehydrated inethanol. The probe hybridization mixture consisted of 50% deionizedformamide, 10% dextran sulfate, 2× SSC (pH 6.0), 0.2 μg/ml probe DNA,and 1 mg/ml sonicated salmon sperm DNA. The probe was denatured in thehybridization solution at 70° C. for 5 minutes. Fifty microliters ofhybridization mix were placed on each slide. Slides were overlaid withcover-slips, sealed with rubber cement, and incubated at 37° C.overnight. Posthybridization washes were three times in 50% deionizedformamide, 2× SSC for 3 minutes and five times for 3 minutes in 2× SSC(pH 7.0) at 39° C. The slides were sequentially dehydrated in ethanol.They were coated with Kodak NTB/2 emulsion, exposed for 3-5 weeks at 4°C., and developed (Harper and Saunders, 1981, supra). Chromosomes werestained with a modified fluorescence, 0.25% Wright's stain procedure(Lin et al., 1995, Cytogenet. Cell Genet. 39:269). The positions ofsilver grains directly over or touching well-banded metaphasechromosomes (FIG. 16) were mapped to an ISCN idiogram (FIG. 17).

The analysis of the distribution of 300 silver grains following in situsublocalization revealed a significant clustering of grains on the shortarm of chromosome 9. 59 silver grains were observed on this region, witha peak distribution at 9p21 (P<0.0001). The assignment of Tek to humanchromosome 9p21 rather than to lp33-34 which is the map location of Tie,demonstrates that during evolution, the region of mouse chromosome 4, towhich both of these RTKs map, has been fragmented and distributed tohuman chromosomes 1 and 9. This is in keeping with earlier datademonstrating that these two regions of the human chromosome are knownto share senteny to mouse chromosome 4.

The human chromosome 9p21 region has been shown to be deleted orrearranged in many types of neoplasia (Fountain et al., 1992; Taguchi etali., 1993; Olopade et al., 1992; Rowley and Diaz, 1992). The latentoncogenic potential of receptor tyrosine kinase proteins and their knownactivation or gene amplification in malignancy suggests that if Tekreceptor tyrosine kinase protein is indeed playing a role in theseneoplasms it is most likely not due to a loss of heterozygosity, but toan activation of the Tek locus. The identification of a new non-randomrearrangement involving (8,9)(q12; p21) in lymphoid malignancies (Huretet al., 1990) suggests that activation of the Tek locus may beresponsible for these or other types of neoplasia.

EXAMPLE XII

The following methods were used in the investigations described inExample XII: Generation of the tek^(A853) Dominant-Negative Transgenesand

Transgenic Embryos

The codon for lysine 853 was altered by oligonucleotide directedmutagenesis (Amersham) to the codon encoding an alanine residue. Theentire cDNA fragment used in this mutagenesis was completely sequencedbefore subcloning back into the full length tek cDNA. The mutated cDNA(tek^(A853)) was cloned into the mammalian expression vector pECE (Elliset al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84:5101-5105) andtransfected into COS cells as described previously. Metabolic labellingand tyrosine kinase assays were done with an anti-Tek antibody asdescribed (Lhotak and Pawson, 1993, Mol. Cell Biol. 13:7071-7079). Twoof the three transgenes were made by cloning the tek^(A853) cDNAupstream of the SV40 polyadenylation (polyA) sequences (BamHI- XbaI) andthen cloning this cassette downstream of the large β-actin promoter(Gift of V. Giguiere, Hospital for Sick Children, Toronto, Canada) orthe 7.2 kb tek promoter. The polyoma-promoter driven transgene wasconstructed by cloning the tek^(A853) cDNA without the SV40 polyAsequences into pdPX₁₃ Bla₃ MT₅ (Bautch et al., 1987, Cell 66:257-270) inwhich the sequences coding for polyoma middle T-antigen had been removedby BstXI digestion. These transgenes all contained 3' untranslatedsequences from the tek cDNA, thus whether transcription terminated atthe tek polyA sequences or the viral polyA sequences is not known. DNAfrom these constructs were prepared and injected into fertilizedoocytes, as previously described (Logan et al., 1993). Embryos wereanalyzed on days 9.5 and 10.5 post-injection and were genotyped by PCRanalysis of yolk sac DNA prepared as described (Frohman et al., 1990),utilizing a primer which annealed within the tek 3' untranslatedsequence (CCTCACCTGCAGAAGCCAGTTTGT) (SEQ ID NO:11) and primers withineither the SV40 (GTGGTTTGTCCAACTCATCAATG) (SEQ ID NO:12) or polyoma(CTACCATAATCCAGTCTACTGC) (SEQ ID NO:13) PolyA sequences. tek TargetingVector

The tek genomic clone used in these studies was obtained from a 129Svmouse strain library. The targeting vector consisted of a long arm 7.2kb Asp718I-BglII genomic fragment located 5' of the tek coding sequencesand a short arm of 0.7 kb extending from XbaI to the EcoRI sitesimmediately 3' of the first exon (see FIG. 20). These two fragments werecloned on either side of the phosphoglycerate kinase (PGK)--neoexpression cassette of the PPNT vector (Tybulewicz et al., 1991, Cell65:1153-1163) such that the direction of neo transcription was in thesame orientation as tek. Upon homologous recombination, this vector willdelete approximately 0.7 kb of genomic sequences which includes 14 bp ofuntranslated sequence, the first 52 nucleotides of the protein-codingsequence and approximately 650 bp of the first intron.

Generation of Transgenic Mice Carrying a Tek cDNA Encoding aDominant-Negative Tek Receptor Tyrosine Kinase Protein

To further assess rapidly the role of the Tek signalling pathway inmouse development, a mutation was introduced within the tek cDNA whichaltered the codon for lysine 853 to encode an alanine residue. Thislysine residue and its surrounding amino acids are found in a regionwithin the intracellular cytoplasmic domain that is highly conserved inall tyrosine kinases and alteration of this residue is known to abolishcatalytic function (Nocka et al., 1990, EMBO J. 9:1805-1813 and; Reithet al., 1990, Genes and Development 4:390-400). Thus, altering lysine⁸⁵³to alanine⁸⁵³ should generate a Tek molecule that is still competent tobind ligand, but which is unable to transduce a signal due to its lackof catalytic function. To determine whether the lysine to alaninemutation at codon 853 affected the intrinsic tyrosine kinase activity ofTek protein, this mutated tek cDNA (tek^(A853)) was introduced into COScells and extracts from these cells were analyzed for Tek activity.

FIG. 18A is a schematic showing the transgenes used to drive theexpression of the dominant-negative mutant tek^(A853) cDNA. The solidbox represents the promoter region for each transgene; the splice donor(SD) and acceptor (SA) of the β-actin promoter are indicated; theImmunoglobulin- (Ig), epidermal growth factor- (EGF), and fibronectintype III-like (FN) repeats found in the extracellular region of Tek aredepicted by cross-hatched, stippled and open boxes, respectively; thesmaller solid box represents the transmembrane region (TM). The twokinase domains (TK1 & TK2) are depicted by open boxes separated by thekinase insert (KI); ovals at the end of each transgene represent thedifferent viral polyadenylation sequences. The oval above TK1 representsthe position of the Lys→Ala853 mutation.

FIG. 18B shows that Tek⁸⁵³ is catalytically inactive. Both thetek^(A853) and wild type tek cDNAs were expressed in COS cells using themammalian expression vector, pECE. Transfected COS cells weremetabolically labelled with [³⁵ S]-methionine and immunoprecipitatedwith anti-Tek antiserum. The immunoprecipitates were split and a portionused in an in vitro kinase assay (left two lanes) while the other waselectrophoresed in a gel similar to the one used to analyze the productsof the kinase assay but after electrophoresis the gel was processed forfluorography (right two lanes). DN, dominant-negative mutant tek^(A853); WT, wild type tek cDNA.

As noted above, Tek^(A853) protein was catalytically inactive inautophosphorylation and phosphorylation of exogenously added substrate(FIG. 18B, and data not shown). Moreover, the engineered mutation didnot alter the length of the protein as judged by its gel mobility (FIG.18B).

The β-actin, polyoma and tek promoters were used to drive expression ofthe tek^(A853) cDNA within the endothelial cell lineage of transgenicmice (FIG. 18A and 18B). The β-actin promoter element is thought to beactive in virtually all cells and thus should drive transgene expressionearly within the endothelial lineage and at relatively high levels.Transgenic animals expressing polyoma middle T-antigen driven by itspromoter succumb to endotheliomas (Bautch et al., 1987, Cell 51:529-538;Williams et al., 1988, Cell 52:121-131) and endothelial cells isolatedfrom these tumors express the transgene (Dubois et al., 1991, Exp. CellRes. 196:302-313). Thus we reasoned that the polyoma early promotersequences would be a good candidate for driving transgene expressionwithin the endothelial cell lineage. Finally, we also employed a 7.2 kbDNA fragment that lies immediately upstream of the tek coding regionwhich we have shown recapitulates the endogenous tek expression profileduring early mouse development.

Tek^(A853) Transgenic Mice are Developmentally Delayed and Exhibit aDefect in Their Endothelium

Based on the assumption that Tek receptor tyrosine kinase may play acritical role in the endothelial cell lineage, transgenic founderembryos were removed on Days 9.5 and 10.5 of gestation, two to threedays after the onset of tek expression. As shown in Table 3, embryostransgenic for the β-actin-tek^(A853) transgene showed no discerniblephenotype. In contrast, two out of 6 transgenic embryos containing thetek-promoter-tek^(A853) transgene were delayed or arrested in theirdevelopment (Table 3 and FIGS. 19A, B and C). In particular, FIGS. 19Aand 19B show that tek^(A853) transgenic mice are developmentally delayedand exhibit a defect in their endothelium. FIG. 19A shows anon-transgenic littermate taken from the same experiment as the embryoin panel B. FIG. 19B shows a tek promoter driven developmentally delayedembryo. FIG. 19C shows a polyoma driven developmentally delayed embryo.All embryos were recovered at E9.5 and were photographed at the samemagnification.

Interestingly, one of the embryos isolated on E9.5 had an enlargedpericardial cavity and contained few blood cells in the vessels of theyolk sac. This was likely due to hemorrhaging into the yolk sac cavity,as primitive red blood cells were observed there. Furthermore, 5 out of19 transgenic polyoma-promoter-tek^(A853) embryos exhibited adevelopmental delay phenotype (Table 3). Of these delayed embryos, twoappeared to have arrested early in development around Day 8.0 as judgedby the closure of their neural folds. The three other embryos weredelayed in their development to varying levels, but appearedmorphologically normal when compared to embryos of the same size. Oneembryo was developmentally arrested, but proved to be negative for thepresence of the transgene by PCR. This embryo was an amorphous masswhich was undergoing resorption suggesting that its development arrestedprior to the onset of tek expression and thus was considered to bephenotypically distinct.

Histological analysis of the developmentally delayed transgenic embryoswas carried out on all tek^(A853) mutants isolated on Day 9.5 (Table 3),but was not performed on Day 10.5 mutants due to severe necrosis of thespecimens. FIGS. 21A-D shows a histological examination of the heartregions from dominant-negative tek^(A853) transgenic and tek.sup.Δspheterozygous and homozygous embryos. E9.5 transgenic embryos, containingthe tek^(A853) transgene driven by either the tek-promoter (FIG. 21A) orthe polyoma early sequences (FIG. 21B). tek.sup.Δsp heterozygous (FIG.21C) and homozygous (FIG. 21D) embryos. tek.sup.Δsp heterozygous embryos(FIG. 21C) showed normal (arrowheads) while mutant transgenictek-promoter-(FIG. 21A) and polyoma-promoter-tek^(A853) (FIG. 21B) andthe tek.sup.Δsp homozygous (FIG. 21D) embryos showed degeneratingendothelium (arrows) within their heart regions. All sections arephotographed at the same magnification . Bar: 10 μm.

The heart of the tek-promoter-tek^(A853) mutant embryos was reduced insize when compared to their normal littermates (FIGS. 21A & C,respectively). The organization of the trabeculae within the heartappeared to be relatively normal; however, there was a reduction in thenumber and complexity of the branching structures (FIG. 21A & C). Theendothelial cells of the endocardium of the heart were fewer in numberand had a short ribbon-like structure which may reflect degeneration.The developmentally delayed embryos observed after microinjection of theployoma-promoter-tek^(A853) transgene manifested phenotypes which variedin their severity. Histological analysis of three of these mutantsrevealed no clear pathological abnormalities, although subtleabnormalities could not be excluded. In contrast, the embryo shown inFIG. 21B represents the most extreme mutant where a defect could clearlybe distinguished. Thin sections of the heart depicted a pathologyvirtually indistinguishable from that observed for tek.sup.Δsp targetedhomozygous mutant embryos (see below). The development and number oftrabeculae within this polyoma-promoter-tek⁸⁵³ and tek.sup.Δsp targetedhomozygous mutant hearts was severely reduced and the extent ofmyocardial development adversely affected (FIGS. 21B & D). Theendothelial cells of the endocardium were few in number and not closelyassociated with the myocardium. In addition, the endothelial cells hadsmall granules on their surfaces, which may be calcium depositsindicating cell death or cellular degeneration. Transgene expressionlevels could not be ascertained by RNA in situ analysis using probesdirected against the viral polyadenylation sequences, suggesting thateither they were not used and that the tek polyadenylation sequenceswithin the tek cDNA were utilized or that the levels were too low to bedetected.

No other overt phenotype was observed for any of the tek^(A853)-dominant-negative embryos, demonstrating that expression of thisprotein in other cellular compartments had no effect. Moreover, the factthat a phenotype was seen with the endothelial specific tek-promoterargues that the observed phenotypes for both the tek- andpolyoma-promoter driven transgenes were intrinsic to a defect in thevascular endothelium.

EXAMPLE XIII

The following methods were used in the investigations described inExample XIII:

Generation and Genotyping of tek.sup.Δsp Mice

R1 (Nagy et al., 1993, Proc. Natl. Acad. Sci. 90:8424-8428) ES cellswere propagated, electroporated, plated and selected as described(Joyner et al., 1989, Nature 338:153-156). Selection in gancyclovirresulted in an enrichment of 7- and 32-fold in the two experiments. Fourtargeted clones were identified (1 in 232 and 3 in 55, respectively).Taken together, the frequency of homologous recombination wasapproximately 1 in 960 G418^(R) clones. The identification of targetedevents was accomplished by Southern blot analysis on ES cell DNAextracted directly in 24 well culture dishes as described (Wurst andJoyner, 1993, "Production of Targeted Embryonic Stem Cell Clones", inGene Targeting, A. L. Joyner, ed. New York, Oxford University Press, pp.33-61) and digested with BglII. A 0.3 kb AccI-BglII genomic DNA fragmentlocated immediately 3' to the short arm was used as probe. This proberecognizes a wild-type fragment of 2.5 kb and a targeted fragment of 1.9kb (see FIG. 20B).

Confirmation of a correctly targeted event was accomplished by Southernanalysis of DNA extracted from heterozygous mice and digested withmultiple enzymes. The probes used were the 3' external and two otherinternal probes consisting of the neo coding sequences and a genomic DNAfragment of 0.4 kb (Spe I-Bgl II) found 5' to the protein codingsequences (data not shown). No non-repetitive probes could be found 5'of the Asp718I site. Injection of ES cells carrying the tek.sup.Δspmutation into C57BL/6J blastocysts was performed as described previously(Joyner et al., 1989, Nature 338:153-156). Genotyping of offspring wascarried out on DNA extracted from either tails or the dissected heads ofembryos. Genotyping of LacZ transgenic animals were determined bySouthern analysis using LacZ coding sequences as probe. Histology andLacZ staining

Midday of the vaginal plug was considered as Day 0.5 post-coitum in thestaging of embryos. To date all embryos with a cobblestone-likeappearing yolk sac were homozygous for the tek.sup.Δsp mutation.Therefore, to conserve material, embryos used in the LacZ-expressionstudies were judged to be homozygous for the tek.sup.Δsp mutation basedon this criteria. Staining for the presence of β-galactosidase inwhole-mount embryos was performed as described (Logan et al., 1993,Development 117:905-916). Stained embryos were postfixed in formalin atroom temperature overnight and processed for wax embedding, sectioned at6 μm and counter-stained with nuclear-fast-red. Quantification of thenumber of LacZ-expressing (blue) endothelial cells was accomplished byselecting a single section of an embryo and counting the number ofendoderm and blue endothelial cells per blood island. Subsequenthistological analysis of these mutants revealed other abnormalitiescharacteristic of homozygous mutants which confirmed the phenotyping.For histological and RNA in situ analysis, the heads of embryos wereremoved for DNA extraction and genotyping prior to fixing the embryosovernight in freshly prepared 4% paraformaldehyde at 4° C. Afterfixation embryos were processed for wax embedding, sectioned at 4-6 μmand either used for RNA in situ analysis or stained withhematoxylin-eosin.

Disruption of the Tek Gene in ES Cells and Germ-line Transmission of theMutation

To create a null allele of tek, the last 52 base pairs of exon-1 weredeleted (FIG. 20A), encoding the first 17 amino acids of Tek protein, byhomologous recombination in ES cells. This deletion removes both thestart of translation and the signal peptide. Therefore, this mutant isreferred to as tek.sup.Δsp. A positive/negative-type targeting vector(Mansour et al., 1993, Development 117:13-28) was engineered by cloning7.2 kb of 5' genomic sequence upstream of a bacterial neomycin (neo)cassette (Tybulewicz et al., 1991, Cell 65:1153-1163) and 0.7 kb of 3'genomic sequences downstream.

In the Figures, FIGS. 20A and B show disruption of the tek locus andSouthern blot analysis of wild type, tek.sup.Δsp heterozygous andhomozygous DNA. FIG. 20A is a schematic showing the strategy used todisrupt the coding sequences of the first exon of the tek gene,generating the mutation tek.sup.Δsp. The closed box represents theprotein-coding sequences; open box represents the untranslatedsequences. The PGK-neo expression cassette, represented by a crossedhatched box, was inserted in the same transcriptional orientation as thetek gene. The stippled box represents the PGK-tk expression cassettefused to plasmid sequences represented by small, open-ended boxes. TheXbaI and EcoRI restriction maps sites are not indicated 5' of the firstexon. The brackets around the 5' Asp718 I site signify that the site wasdestroyed as a consequence of cloning. The location of the 3' externalprobe is indicated by a closed box beneath the predicted targeted locus.

FIG. 20B DNA extracted from Day 9.5 embryos from a tek.sup.Δsp/+heterozygous F₁ intercross. The presence of the tek.sup.Δsp specificfragment is indicated, Trg. The number of wild type (+/+), heterozygous(+/-) and homozygous (-/-) embryos (2, 4 and 3, respectively) were atthe predicted Mendelian frequency.

In two separate experiments, linearized targeting vector waselectroporated into RI ES cells as described above. A properly targetedevent was observed (FIG. 20B and data not shown) by Southern blot withboth a 3' external and internal probes. Two independent ES cell linescarrying the tek.sup.Δsp allele were injected into host C57BL/6Jblastocysts to generate Chimeras that transmitted the mutation to theiroffspring.

tek.sup.Δsp Homozygous Mice Die During Gestation

Mice heterozygous for the tek.sup.Δsp mutation had no apparentabnormalities and were fertile. Intercrosses of mice derived from bothindependent ES cell clones were carried out between either outbred(129SvJ×C57BL/6J) F₁ or inbred 129SvJ F₁ mice to allow analysis on twogenetic backgrounds. No differences in phenotype were observed on eitherof the two genetic backgrounds or the two targeted ES cell lines.

F₁ intercrosses of tek.sup.Δsp /+mice produced no live offspringhomozygous for the tek.sup.Δsp allele (Table 4). Mothers from theseintercrosses were therefore sacrificed and embryos were genotyped. AtE9.5 some embryos from the heterozygous cross were visibly defective,showing some signs of necrosis and their hearts were not beating. Theseembryos were all homozygous for the teksP mutation (Table 4) No livehomozygous mutant embryos were found beyond E9.5 (Table 4). At E12.5,none of the embryos (0/35) were teksP homozygotes; however, there were 8severely necrosed implantations, suggesting that tek.sup.Δsp homozygousembryos implanted, but then died. Genotyping of embryos isolated on E9.5demonstrated that the proportion of embryos that were wild type,heterozygous and homozygous for the tepsP allele followed the expectedMendelian frequency, confirming that Tek is not required forimplantation of the embryo (FIG. 20B and Table 4).

Hemorrhaging of tek.sup.Δsp /tek.sup.Δsp Embryos

FIGS. 22A-F shows a histological analysis of homozygous tek mutantembryos and normal littermates. In particular, the Figures show Sectionsthrough the embryonic portion of the placenta from tek.sup.Δspheterozygous (22A) and homozygous (22D) embryos showing the accumulationof fetal blood cells in the placental sinuses in homozygous embryos.These sections also illustrate the decreased number of endothelial cellsin the sinus of mutants as compared to normal littermates (arrowheads).Bar: 10 μm. Thin sections taken through the dorsal aortic region ofheterozygous (22B) and homozygous (22E) embryos showing the collapsedaorta (da) and extravasated blood (arrows). Bar: 30 μm. Stained thinsections through the yolk sac of tek.sup.Δsp heterozygous (22C) andhomozygous (22F) embryos showing the distended yolk sac vessels and thedecreased number of endothelial cells lining the yolk sac vessels(arrowheads). Bars: 30 μm.

FIG. 23A-D shows the yolk sac vasculature of tek.sup.Δsp homozygousembryos contain fewer endothelial cells. tek-promoter-lacz transgeneexpression in Day 8.5 normal (23A) and tek.sup.Δsp homozygous (23C)embryos shows a reduced number of blue staining endothelial cells in thehomozygous mutants. The decreased number of blue cells (arrowheads) iseven more dramatic in the yolk sac of Day 9.0 tek.sup.Δsp homozygous(23D) embryos as compared to normal embryos (23B). Bar:50 μm.

FIGS. 24A-D shows the embryonic vasculature of tek.sup.Δsp homozygousembryos contain fewer endothelial cells. The trunk (24A, 24C) and heart(24B, 24D) regions of a E9.0 tek.sup.Δsp homozygous (24A, 24B) and wildtype (24C, 24D) embryos. A lower levels of lacZ expression is seen inthe intersegmental vessels (is) and endocardium (e) of mutants. Dorsalaorta, da. Bars: 50 μm.

In summary, the data show that Day 8.5 embryos homozygous for thetek.sup.Δsp mutation were readily discernible by the grossly abnormalmorphology of their yolk sacs, which were engorged with blood and had acobble-stone-like appearance (FIG. 22F & 23A-D). To date all embryoswith this morphologically distinct yolk sac that have been genotypedhave been homozygous tek.sup.Δsp (9/9).

The histological analysis of the yolk sacs from wild-type orheterozygous embryos harvested on E9.5 revealed that the blood vesselsin the yolk sac appeared distended (FIGS. 23C & D) and were very oftenpacked with blood (FIG. 24C & D). In contrast, several yolk sacsisolated from homozygous embryos contained little or no blood (FIG.22F). Prior to dissection of these embryos, however, blood could bedetected in the yolk sac cavity, indicating that the lack of blood inthe yolk sac vasculature was due to hemorrhaging. In addition, the yolksac vessels contained considerably fewer endothelial cells (FIG. 22F)than heterozygous littermates (FIG. 22C). Furthermore, vascularhemorrhaging of homozygous embryos could also be detected histologicallywhen the trunk region was examined. Primitive blood cells could be seenthroughout the body of the embryo distributed among the mesenchymalcells (FIGS. 22B & E). The dorsal aorta in heterozygous embryos was welldefined with endothelial cells lining the lumen of the vessel and therewas no blood in the trunk (FIG. 22B). In contrast, in homozygous embryosthe endothelium of the dorsal aorta was disorganized and appeared tohave ruptured, resulting in blood cells in the body (FIG. 22E).Localized hemorrhaging of the embryonic vasculature most likely resultsin a decrease in the embryonic blood pressure which may explain theaccumulation of blood in the yolk sac vasculature and embryonic portionof the placenta (FIG. 22D). This region of the placenta also had veryfew endothelial cells in the sinuses as compared to a heterozygouslittermate (FIGS. 22A & D). These results clearly demonstrate thattek.sup.Δsp /tek.sup.Δsp embryos have a striking deficiency in theendothelium, resulting in hemorrhaging and pooling of blood in bodycavities.

The hearts of tek.sup.Δsp homozygous embryos were severelyunder-developed (FIG. 21D). The myocardium of E9.5 mutant embryos didnot possess a detailed organization of trabeculae and the overall growthof the myocardium seems to be reduced. Furthermore, fewer endothelialcells were seen in the endocardium (FIG. 21D).

Analysis of flk-1, tek, and tie expression in tek.sup.Δsp embryos

That there were few remaining endothelial cells in homozygous embryoswas confirmed by RNA in situ hybridization of sections prepared fromboth tek.sup.Δsp homozygous and heterozygous embryos with a flk-1antisense riboprobe. Both heterozygous and homozygous embryos (data notshown) contained flk-1-positive cells organized in a distinctivevascular network. However, the flk-1 positive cells in homozygous mutantembryos were present in discontinuous chains, suggesting that thevessels contained a sparsely populated endothelium (data not shown).Moreover the levels of flk-1 expression were lower in the homozygousmutants. Adjacent sections probed for the expression of tek and tiedemonstrated that tie transcripts were present albeit at lower levelsthan in heterozygous embryos (data not shown), whereas no tek signalscould be detected in homozygous tek.sup.Δsp embryos (data not shown).These results demonstrate that the tek.sup.Δsp mutant allele does notproduce a normal transcript, confirming that it is a null allele. Veryinterestingly, these results also demonstrate that tie expression inendothelial cells is not dependent on prior expression of tek.

tek.sup.Δsp /tek.sup.Δsp Embryos Have a Reduced Number of EndothelialCells

In order to follow the fate of tek expressing endothelial cells inmutant embryos, a tek-promoter-LacZ transgene gene was crossed onto thetek.sup.Δsp mutant background. Adult mice bearing the tek-promoter-lacZ,tek.sup.Δsp /+ genotype were then used to generate homozygous embryoscarrying the tek.sup.Δsp mutation and the transgene. Thetek-promoter-lacZ transgenic line used in these studies expresses theLacZ reporter gene in a manner which virtually recapitulates theendogenous tek expression profile.

Based on β-galactosidase (β-gal) activity, tek.sup.Δsp homozygousembryos isolated on E8.5 and E9.0 contained a normally patternedvasculature in both extra- and embryonic tissues (FIGS. 24A-D and datanot shown). Moreover, the size of normal and homozygous embryos at thesegestational ages were the same (FIGS. 24A-D and data not shown),suggesting that the growth of the embryo up to E9.0 is not dependent onTek. However, it is clear that the level of β-gal staining in thesehomozygous embryos was reduced (FIGS. 24A-D and 25A-D, and data notshown). Histological examination of E9.0 homozygous embryos confirmedthat proper patterning of the vasculature was initiated (FIGS. 24A & B,and data not shown). Furthermore, the endocardium and other vascularstructures of mutant embryos formed correctly but contained only lowlevels of LacZ expression, in keeping with the low levels of flk-1 andtie expression detected in these cells (FIG. 24A-D and data not shown).

FIGS. 25A-D show endothelial cells in the yolk sac of tek.sup.Δsphomozygous embryos express low levels of the tek-lacZ transgene. Thinsections taken from the yolk sacs presented in FIGS. 25A-D illustratetek-promoter-lacz expression (arrowheads) in the endothelial cells ofE8.5 (25A,C) and E9.0 (25B,D) tek.sup.Δsp homozygous (25C,D) and wildtype (25A,B) embryos. These photomicrographs show both a reduction inthe number of blue staining endothelial cells and a decrease in thelevels of β-Galactosidase activity in the mutants. In addition,increased blood cell number can be seen in the blood vessels oftek.sup.Δsp homozygous embryos. Bars: A&C=25 μm; B&D=12.5 μm.

As shown in the Figures, histological analysis of the yolk sacs oftek.sup.Δsp homozygous mutants revealed that the number of lacZexpressing endothelial cells lining the blood islands was reduced inE8.5 tek.sup.Δsp homozygous embryos (FIG. 25C) compared to their normallittermates (FIG. 25A). This decrease in cell number and stainingintensity was even more accentuated in sections taken from E9.0homozygous embryos (FIGS. 25B & D). The blood islands also containedcells with an endothelial cell-like morphology which did not stain blue,whereas this was never observed in normal transgenic mice.

Table 5 summarizes the number of blue endothelial cells found in theyolk sacs of transgenic embryos. The number of endoderm cells found ineach blood island did not vary significantly for any of the embryos andthus was used to normalize the values. Day 8.5 tek.sup.Δsp homozygousembryos possessed approximately 30% fewer endothelial cells within theblood islands as compared to their normal littermates. On E9.0, one halfday later in development, 75% fewer endothelial cells were detected inthe yolk sac of tek.sup.Δsp homozygous mutant embryos. These resultsclearly demonstrate that the number of endothelial cells present withinhomozygous embryos at the times analyzed is significantly lower thanthat of their normal littermates and that as development progresses thenumber of endothelial cells decreases. Moreover, the very low levels ofLacZ expression detected in many cells suggests that these cells areprobably compromised metabolically and are dying.

                  TABLE 1                                                         ______________________________________                                        Protein tyrosine kinase cDNAs isolated by RT-PCR                                  Embryonic Age                                                                            cDNA                                                           (Days)     tek     pdgfrb  c-abl  c-src                                                                              bmk                                    ______________________________________                                        9.5        26      7       2      1    1                                        12.5 5 10 --  -- --                                                         ______________________________________                                    

                                      TABLE 2                                     __________________________________________________________________________    Cosegregation of the tek, brown, and pmv-23 loci in A × D strains.       A × D strain                                                           Locus                                                                             1 2 3 6 7 8 9 10                                                                              11                                                                              12                                                                              13                                                                              14                                                                              15                                                                              16                                                                              18                                                                              20                                                                              21                                                                              22                                                                              23                                                                              24                                                                              25                                                                              26                                                                              27                                                                              28                          __________________________________________________________________________    tek D D A D D A A A D A D A D D D D A D D A D D D D                             brown D D A D D A A A D A A A D D D D A D A A D D D D                         pmv-23 D D A D D A D A D A D D D A D D A D D A D D D A                      __________________________________________________________________________

                  TABLE 3                                                         ______________________________________                                        Delayed development among Tek.sup.A853                                          Dominant-Negative Transgenic Embryos                                                      Total    Total   Total Devel                                       Embryos Transgenic Delayed                                                   Transgene Recovered (TG) (DD) DD-TG/DD                                      ______________________________________                                        Polyoma-tek.sup.A853                                                                    126      19        6.sup.# 5*/6                                       tek-tek.sup.A853 64 6 2.sup.#  2/2                                            β-actin-tek.sup.A853 20 6 0  --                                        ______________________________________                                         *One embryo comprised a small amorphous mass of necrotic cells that was       undergoing resorption at the time of assay. As such, it was considered to     be phenolypically distinct from the group of transgenic embryos showing       the TeK.sup.A853 dominant negative phenotype.                                 .sup.# All embryos were obtained for analysis on E9.5 except that two wer     discovered with the polyoma driven transgene on E10.5 and 1 with the          tekpromoter on E10.5.                                                    

                  TABLE 4                                                         ______________________________________                                        Genotypes of progeny of F.sub.1 intercrosses of tek.sup.Δsp /+          heterzygous mice                                                                       Genotypes                                                                   neonates     E9.5                                                      Clone    +/+    +/-       -/- +/+     +/- -/-                                 ______________________________________                                        24       108    57        0   9       6   7                                     19 11 4 0 3 1 1                                                               Total 119 61 0 12 7 8                                                       ______________________________________                                         Genotyping was carried out by Southern analysis on DNA extracted from         tails or from the dissected head of embryos                              

                  TABLE 5                                                         ______________________________________                                        The ratio of endoderm cells to LacZ-positive cells in the yolk sacs of         embryos of F1 intercrosses of tek-LacZ/tek-LacZ; tek.sup.Δsp /+        mice.                                                                           Gest-           Total number                                                                            Total number                                                                            Number of                                 ational tek of endoderm of LacZ.sup.+ LacZ.sup.+  cells                       Age Geno- cells per expressing cells per 100                                  (Days) type blood island.sup.# per blood island endoderm cells              ______________________________________                                        8.5   +/-     7.8 ± 0.9 (24)                                                                         4.5 ± 0.7                                                                             54 ± 12                                 8.5 -/- 6.1 ± 1.1 (45) 1.0 ± 0.8 35 ± 7                              9.0 +/- 11.8 ± 0.3 (27)  4.7 ± 0.6 39 ± 6                            9.0 -/- 11.8 ± 3 (21)   1.1 ± 0.9  8 ± 4                           ______________________________________                                         .sup.# Numbers reflect the mean ± S.D. and the number in brackets          represents the number of blood islands counted per section.              

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 32                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4175 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -      (v) FRAGMENT TYPE: N-terminal                                        - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Mus muscu - #lus                                                (B) STRAIN: CD-1                                                              (D) DEVELOPMENTAL STAGE: - #Embryo                                            (F) TISSUE TYPE: Heart                                               - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: Tek                                                       - -   (viii) POSITION IN GENOME:                                                       (A) CHROMOSOME/SEGMENT: 4                                                     (B) MAP POSITION: Betwe - #en the brown and pmv-23 loci             - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 124..3478                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - GCCAACTTGT AAACAAGAGC GAGTGGACCA TGCGAGCGGG AAGTCGCAAA GT -             #TGTGAGTT     60                                                                 - - GTTGAAAGCT TCCCAGGGAC TCATGCTCAT CTGTGGACGC TGGATGGGGA GA -            #TCTGGGGA    120                                                                 - - AGT ATG GAC TCT TTA GCC GGC TTA GTT CTC TG - #T GGA GTC AGC TTG        CTC      168                                                                        Met Asp Ser Leu Ala Gly Leu Val - #Leu Cys Gly Val Ser Leu Leu                 1            - #   5               - #   10               - #   15       - - CTT TAT GGA GTA GTA GAA GGC GCC ATG GAC CT - #G ATC TTG ATC AAT TCC          216                                                                       Leu Tyr Gly Val Val Glu Gly Ala Met Asp Le - #u Ile Leu Ile Asn Ser                            20 - #                 25 - #                 30              - - CTA CCT CTT GTG TCT GAT GCC GAA ACA TCC CT - #C ACC TGC ATT GCC TCT          264                                                                       Leu Pro Leu Val Ser Asp Ala Glu Thr Ser Le - #u Thr Cys Ile Ala Ser                        35     - #             40     - #             45                  - - GGG TGG CAC CCC CAT GAG CCC ATC ACC ATA GG - #A AGG GAC TTT GAA GCC          312                                                                       Gly Trp His Pro His Glu Pro Ile Thr Ile Gl - #y Arg Asp Phe Glu Ala                    50         - #         55         - #         60                      - - TTA ATG AAC CAG CAC CAA GAT CCA CTG GAG GT - #T ACT CAA GAT GTG ACC          360                                                                       Leu Met Asn Gln His Gln Asp Pro Leu Glu Va - #l Thr Gln Asp Val Thr                65             - #     70             - #     75                          - - AGA GAA TGG GCG AAA AAA GTT GTT TGG AAG AG - #A GAA AAG GCC AGT AAG          408                                                                       Arg Glu Trp Ala Lys Lys Val Val Trp Lys Ar - #g Glu Lys Ala Ser Lys            80                 - # 85                 - # 90                 - # 95       - - ATT AAT GGT GCT TAT TTC TGT GAA GGT CGA GT - #T CGA GGA CAG GCT ATA          456                                                                       Ile Asn Gly Ala Tyr Phe Cys Glu Gly Arg Va - #l Arg Gly Gln Ala Ile                           100  - #               105  - #               110              - - AGG ATA CGG ACC ATG AAG ATG CGT CAA CAA GC - #A TCC TTC CTA CCT GCT          504                                                                       Arg Ile Arg Thr Met Lys Met Arg Gln Gln Al - #a Ser Phe Leu Pro Ala                       115      - #           120      - #           125                  - - ACT TTA ACT ATG ACC GTG GAC AGG GGA GAT AA - #T GTG AAC ATA TCT TTC          552                                                                       Thr Leu Thr Met Thr Val Asp Arg Gly Asp As - #n Val Asn Ile Ser Phe                   130          - #       135          - #       140                      - - AAA AAG GTG TTA ATT AAA GAA GAA GAT GCA GT - #G ATT TAC AAA AAT GGC          600                                                                       Lys Lys Val Leu Ile Lys Glu Glu Asp Ala Va - #l Ile Tyr Lys Asn Gly               145              - #   150              - #   155                          - - TCC TTC ATC CAC TCA GTG CCC CGG CAT GAA GT - #A CCT GAT ATT TTA GAA          648                                                                       Ser Phe Ile His Ser Val Pro Arg His Glu Va - #l Pro Asp Ile Leu Glu           160                 1 - #65                 1 - #70                 1 -      #75                                                                              - - GTT CAC TTG CCG CAT GCT CAG CCC CAG GAT GC - #T GGT GTG TAC TCG        GCC      696                                                                    Val His Leu Pro His Ala Gln Pro Gln Asp Al - #a Gly Val Tyr Ser Ala                          180  - #               185  - #               190              - - AGG TAC ATA GGA GGA AAC CTG TTC ACC TCA GC - #C TTC ACC AGG CTG ATT          744                                                                       Arg Tyr Ile Gly Gly Asn Leu Phe Thr Ser Al - #a Phe Thr Arg Leu Ile                       195      - #           200      - #           205                  - - GTT CGG AGA TGT GAA GCT CAG AAG TGG GGG CC - #C GAC TGT AGC CGT CCT          792                                                                       Val Arg Arg Cys Glu Ala Gln Lys Trp Gly Pr - #o Asp Cys Ser Arg Pro                   210          - #       215          - #       220                      - - TGT ACT ACT TGC AAG AAC AAT GGA GTC TGC CA - #T GAA GAT ACC GGG GAA          840                                                                       Cys Thr Thr Cys Lys Asn Asn Gly Val Cys Hi - #s Glu Asp Thr Gly Glu               225              - #   230              - #   235                          - - TGC ATT TGC CCT CCT GGG TTT ATG GGG AGA AC - #A TGT GAG AAA GCT TGT          888                                                                       Cys Ile Cys Pro Pro Gly Phe Met Gly Arg Th - #r Cys Glu Lys Ala Cys           240                 2 - #45                 2 - #50                 2 -      #55                                                                              - - GAG CCG CAC ACA TTT GGC AGG ACC TGT AAA GA - #A AGG TGT AGT GGA        CCA      936                                                                    Glu Pro His Thr Phe Gly Arg Thr Cys Lys Gl - #u Arg Cys Ser Gly Pro                          260  - #               265  - #               270              - - GAA GGA TGC AAG TCT TAT GTG TTC TGT CTC CC - #A GAC CCT TAC GGG TGT          984                                                                       Glu Gly Cys Lys Ser Tyr Val Phe Cys Leu Pr - #o Asp Pro Tyr Gly Cys                       275      - #           280      - #           285                  - - TCC TGT GCC ACA GGC TGG AGG GGG TTG CAG TG - #C AAT GAA GCA TGC CCA         1032                                                                       Ser Cys Ala Thr Gly Trp Arg Gly Leu Gln Cy - #s Asn Glu Ala Cys Pro                   290          - #       295          - #       300                      - - TCT GGT TAC TAC GGA CCA GAC TGT AAG CTC AG - #G TGC CAC TGT ACC AAT         1080                                                                       Ser Gly Tyr Tyr Gly Pro Asp Cys Lys Leu Ar - #g Cys His Cys Thr Asn               305              - #   310              - #   315                          - - GAA GAG ATA TGT GAT CGG TTC CAA GGA TGC CT - #C TGC TCT CAA GGA TGG         1128                                                                       Glu Glu Ile Cys Asp Arg Phe Gln Gly Cys Le - #u Cys Ser Gln Gly Trp           320                 3 - #25                 3 - #30                 3 -      #35                                                                              - - CAA GGG CTG CAG TGT GAG AAA GAA GGC AGG CC - #A AGG ATG ACT CCA        CAG     1176                                                                    Gln Gly Leu Gln Cys Glu Lys Glu Gly Arg Pr - #o Arg Met Thr Pro Gln                          340  - #               345  - #               350              - - ATA GAG GAT TTG CCA GAT CAC ATT GAA GTA AA - #C AGT GGA AAA TTT AAC         1224                                                                       Ile Glu Asp Leu Pro Asp His Ile Glu Val As - #n Ser Gly Lys Phe Asn                       355      - #           360      - #           365                  - - CCC ATC TGC AAA GCC TCT GGG TGG CCA CTA CC - #T ACT AGT GAA GAA ATG         1272                                                                       Pro Ile Cys Lys Ala Ser Gly Trp Pro Leu Pr - #o Thr Ser Glu Glu Met                   370          - #       375          - #       380                      - - ACC CTA GTG AAG CCA GAT GGG ACA GTG CTC CA - #A CCA AAT GAC TTC AAC         1320                                                                       Thr Leu Val Lys Pro Asp Gly Thr Val Leu Gl - #n Pro Asn Asp Phe Asn               385              - #   390              - #   395                          - - TAT ACA GAT CGT TTC TCA GTG GCC ATA TTC AC - #T GTC AAC CGA GTC TTA         1368                                                                       Tyr Thr Asp Arg Phe Ser Val Ala Ile Phe Th - #r Val Asn Arg Val Leu           400                 4 - #05                 4 - #10                 4 -      #15                                                                              - - CCT CCT GAC TCA GGA GTC TGG GTC TGC AGT GT - #G AAC ACA GTG GCT        GGG     1416                                                                    Pro Pro Asp Ser Gly Val Trp Val Cys Ser Va - #l Asn Thr Val Ala Gly                          420  - #               425  - #               430              - - ATG GTG GAA AAG CCT TTC AAC ATT TCC GTC AA - #A GTT CTT CCA GAG CCC         1464                                                                       Met Val Glu Lys Pro Phe Asn Ile Ser Val Ly - #s Val Leu Pro Glu Pro                       435      - #           440      - #           445                  - - CTG CAC GCC CCA AAT GTG ATT GAC ACT GGA CA - #T AAC TTT GCT ATC ATC         1512                                                                       Leu His Ala Pro Asn Val Ile Asp Thr Gly Hi - #s Asn Phe Ala Ile Ile                   450          - #       455          - #       460                      - - AAT ATC AGC TCT GAG CCT TAC TTT GGG GAT GG - #A CCC ATC AAA TCC AAG         1560                                                                       Asn Ile Ser Ser Glu Pro Tyr Phe Gly Asp Gl - #y Pro Ile Lys Ser Lys               465              - #   470              - #   475                          - - AAG CTT TTC TAT AAA CCT GTC AAT CAG GCC TG - #G AAA TAC ATT GAA GTG         1608                                                                       Lys Leu Phe Tyr Lys Pro Val Asn Gln Ala Tr - #p Lys Tyr Ile Glu Val           480                 4 - #85                 4 - #90                 4 -      #95                                                                              - - ACG AAT GAG ATT TTC ACT CTC AAC TAC TTG GA - #G CCG CGG ACT GAC        TAC     1656                                                                    Thr Asn Glu Ile Phe Thr Leu Asn Tyr Leu Gl - #u Pro Arg Thr Asp Tyr                          500  - #               505  - #               510              - - GAG CTG TGT GTG CAG CTG GCC CGT CCT GGA GA - #G GGT GGA GAA GGG CAT         1704                                                                       Glu Leu Cys Val Gln Leu Ala Arg Pro Gly Gl - #u Gly Gly Glu Gly His                       515      - #           520      - #           525                  - - CCT GGG CCT GTG AGA CGA TTT ACA ACA GCG TG - #T ATC GGA CTC CCT CCT         1752                                                                       Pro Gly Pro Val Arg Arg Phe Thr Thr Ala Cy - #s Ile Gly Leu Pro Pro                   530          - #       535          - #       540                      - - CCA AGA GGT CTC AGT CTC CTG CCA AAA AGC CA - #G ACA GCT CTA AAT TTG         1800                                                                       Pro Arg Gly Leu Ser Leu Leu Pro Lys Ser Gl - #n Thr Ala Leu Asn Leu               545              - #   550              - #   555                          - - ACT TGG CAA CCG ATA TTT ACA AAC TCA GAA GA - #T GAA TTT TAT GTG GAA         1848                                                                       Thr Trp Gln Pro Ile Phe Thr Asn Ser Glu As - #p Glu Phe Tyr Val Glu           560                 5 - #65                 5 - #70                 5 -      #75                                                                              - - GTC GAG AGG CGA TCC CTG CAA ACA ACA AGT GA - #T CAG CAG AAC ATC        AAA     1896                                                                    Val Glu Arg Arg Ser Leu Gln Thr Thr Ser As - #p Gln Gln Asn Ile Lys                          580  - #               585  - #               590              - - GTG CCT GGG AAC CTG ACC TCG GTG CTA CTG AG - #C AAC TTA GTC CCC AGG         1944                                                                       Val Pro Gly Asn Leu Thr Ser Val Leu Leu Se - #r Asn Leu Val Pro Arg                       595      - #           600      - #           605                  - - GAG CAG TAC ACA GTC CGA GCT AGA GTC AAC AC - #C AAG GCG CAG GGG GAG         1992                                                                       Glu Gln Tyr Thr Val Arg Ala Arg Val Asn Th - #r Lys Ala Gln Gly Glu                   610          - #       615          - #       620                      - - TGG AGT GAA GAA CTC AGG GCC TGG ACC CTT AG - #T GAC ATT CTC CCT CCT         2040                                                                       Trp Ser Glu Glu Leu Arg Ala Trp Thr Leu Se - #r Asp Ile Leu Pro Pro               625              - #   630              - #   635                          - - CAA CCA GAA AAC ATC AAG ATC TCC AAC ATC AC - #T GAC TCC ACA GCT ATG         2088                                                                       Gln Pro Glu Asn Ile Lys Ile Ser Asn Ile Th - #r Asp Ser Thr Ala Met           640                 6 - #45                 6 - #50                 6 -      #55                                                                              - - GTT TCT TGG ACA ATA GTG GAT GGC TAT TCG AT - #T TCT TCC ATC ATC        ATC     2136                                                                    Val Ser Trp Thr Ile Val Asp Gly Tyr Ser Il - #e Ser Ser Ile Ile Ile                          660  - #               665  - #               670              - - CGG TAT AAG GTT CAG GGC AAA AAT GAA GAC CA - #G CAC ATT GAT GTG AAG         2184                                                                       Arg Tyr Lys Val Gln Gly Lys Asn Glu Asp Gl - #n His Ile Asp Val Lys                       675      - #           680      - #           685                  - - ATC AAG AAT GCT ACC GTT ACT CAG TAC CAG CT - #C AAG GGC CTA GAG CCA         2232                                                                       Ile Lys Asn Ala Thr Val Thr Gln Tyr Gln Le - #u Lys Gly Leu Glu Pro                   690          - #       695          - #       700                      - - GAG ACT ACA TAC CAT GTG GAT ATT TTT GCT GA - #G AAC AAC ATA GGA TCA         2280                                                                       Glu Thr Thr Tyr His Val Asp Ile Phe Ala Gl - #u Asn Asn Ile Gly Ser               705              - #   710              - #   715                          - - AGC AAC CCA GCC TTT TCT CAT GAA CTG AGG AC - #G CTT CCA CAT TCC CCA         2328                                                                       Ser Asn Pro Ala Phe Ser His Glu Leu Arg Th - #r Leu Pro His Ser Pro           720                 7 - #25                 7 - #30                 7 -      #35                                                                              - - GGC TCT GCA GAC CTC GGA GGG GGA AAG ATG CT - #A CTC ATA GCC ATC        CTT     2376                                                                    Gly Ser Ala Asp Leu Gly Gly Gly Lys Met Le - #u Leu Ile Ala Ile Leu                          740  - #               745  - #               750              - - GGG TCG GCT GGA ATG ACT TGC ATC ACC GTG CT - #G TTG GCG TTT CTG ATT         2424                                                                       Gly Ser Ala Gly Met Thr Cys Ile Thr Val Le - #u Leu Ala Phe Leu Ile                       755      - #           760      - #           765                  - - ATG TTG CAA CTG AAG AGA GCA AAT GTC CAA AG - #G AGA ATG GCT CAG GCA         2472                                                                       Met Leu Gln Leu Lys Arg Ala Asn Val Gln Ar - #g Arg Met Ala Gln Ala                   770          - #       775          - #       780                      - - TTC CAG AAC AGA GAA GAA CCA GCT GTG CAG TT - #T AAC TCA GGA ACT CTG         2520                                                                       Phe Gln Asn Arg Glu Glu Pro Ala Val Gln Ph - #e Asn Ser Gly Thr Leu               785              - #   790              - #   795                          - - GCC CTT AAC AGG AAG GCC AAA AAC AAT CCA GA - #T CCC ACA ATT TAT CCT         2568                                                                       Ala Leu Asn Arg Lys Ala Lys Asn Asn Pro As - #p Pro Thr Ile Tyr Pro           800                 8 - #05                 8 - #10                 8 -      #15                                                                              - - GTG CTT GAC TGG AAT GAC ATC AAG ATC GGA GA - #G GGC AAC TTT GGC        CAG     2616                                                                    Val Leu Asp Trp Asn Asp Ile Lys Ile Gly Gl - #u Gly Asn Phe Gly Gln                          820  - #               825  - #               830              - - GTT CTG AAG GCA CGC ATC AAG AAG GAT GGG TT - #A CGG ATG GAT GCC GCC         2664                                                                       Val Leu Lys Ala Arg Ile Lys Lys Asp Gly Le - #u Arg Met Asp Ala Ala                       835      - #           840      - #           845                  - - ATC AAG AGG ATG AAA GAG TAT GCC TCC AAA GA - #T GAT CAC AGG GAC TTC         2712                                                                       Ile Lys Arg Met Lys Glu Tyr Ala Ser Lys As - #p Asp His Arg Asp Phe                   850          - #       855          - #       860                      - - GCA GGA GAA CTG GAG GTT CTT TGT AAA CTT GG - #A CAC CAT CCA AAC ATC         2760                                                                       Ala Gly Glu Leu Glu Val Leu Cys Lys Leu Gl - #y His His Pro Asn Ile               865              - #   870              - #   875                          - - ATT AAT CTC TTG GGA GCA TGT GAA CAC CGA GG - #C TAT TTG TAC CTA GCT         2808                                                                       Ile Asn Leu Leu Gly Ala Cys Glu His Arg Gl - #y Tyr Leu Tyr Leu Ala           880                 8 - #85                 8 - #90                 8 -      #95                                                                              - - ATT GAG TAT GCC CCG CAT GGA AAC CTC CTG GA - #C TTC CTG CGT AAG        AGC     2856                                                                    Ile Glu Tyr Ala Pro His Gly Asn Leu Leu As - #p Phe Leu Arg Lys Ser                          900  - #               905  - #               910              - - AGA GTG CTA GAG ACA GAC CCT GCT TTT GCC AT - #C GCC AAC AGT ACA GCT         2904                                                                       Arg Val Leu Glu Thr Asp Pro Ala Phe Ala Il - #e Ala Asn Ser Thr Ala                       915      - #           920      - #           925                  - - TCC ACA CTG TCC TCC CAA CAG CTT CTT CAT TT - #T GCT GCA GAT GTG GCC         2952                                                                       Ser Thr Leu Ser Ser Gln Gln Leu Leu His Ph - #e Ala Ala Asp Val Ala                   930          - #       935          - #       940                      - - CGG GGG ATG GAC TAC TTG AGC CAG AAA CAG TT - #T ATC CAC AGG GAC CTG         3000                                                                       Arg Gly Met Asp Tyr Leu Ser Gln Lys Gln Ph - #e Ile His Arg Asp Leu               945              - #   950              - #   955                          - - GCT GCC AGA AAC ATT TTA GTT GGT GAA AAC TA - #C ATA GCC AAA ATA GCA         3048                                                                       Ala Ala Arg Asn Ile Leu Val Gly Glu Asn Ty - #r Ile Ala Lys Ile Ala           960                 9 - #65                 9 - #70                 9 -      #75                                                                              - - GAT TTT GGA TTG TCA CGA GGT CAA GAA GTG TA - #T GTG AAA AAG ACA        ATG     3096                                                                    Asp Phe Gly Leu Ser Arg Gly Gln Glu Val Ty - #r Val Lys Lys Thr Met                          980  - #               985  - #               990              - - GGA AGG CTC CCA GTG CGT TGG ATG GCA ATC GA - #A TCA CTG AAC TAT AGT         3144                                                                       Gly Arg Leu Pro Val Arg Trp Met Ala Ile Gl - #u Ser Leu Asn Tyr Ser                       995      - #           1000      - #          1005                 - - GTC TAT ACA ACC AAC AGT GAT GTC TGG TCC TA - #T GGT GTA TTG CTC TGG         3192                                                                       Val Tyr Thr Thr Asn Ser Asp Val Trp Ser Ty - #r Gly Val Leu Leu Trp                   1010         - #       1015          - #      1020                     - - GAG ATT GTT AGC TTA GGA GGC ACC CCC TAC TG - #C GGC ATG ACG TGC GCG         3240                                                                       Glu Ile Val Ser Leu Gly Gly Thr Pro Tyr Cy - #s Gly Met Thr Cys Ala               1025             - #   1030              - #  1035                         - - GAG CTC TAT GAG AAG CTA CCC CAG GGC TAC AG - #G CTG GAG AAG CCC CTG         3288                                                                       Glu Leu Tyr Glu Lys Leu Pro Gln Gly Tyr Ar - #g Leu Glu Lys Pro Leu           1040                1045 - #                1050 - #               1055        - - AAC TGT GAT GAT GAG GTG TAT GAT CTA ATG AG - #A CAG TGC TGG AGG GAG         3336                                                                       Asn Cys Asp Asp Glu Val Tyr Asp Leu Met Ar - #g Gln Cys Trp Arg Glu                           1060 - #               1065  - #              1070             - - AAG CCT TAT GAG AGA CCA TCA TTT GCC CAG AT - #A TTG GTG TCC TTA AAC         3384                                                                       Lys Pro Tyr Glu Arg Pro Ser Phe Ala Gln Il - #e Leu Val Ser Leu Asn                       1075     - #           1080      - #          1085                 - - AGG ATG CTG GAA GAA CGG AAG ACA TAC GTG AA - #C ACC ACA CTG TAT GAG         3432                                                                       Arg Met Leu Glu Glu Arg Lys Thr Tyr Val As - #n Thr Thr Leu Tyr Glu                   1090         - #       1095          - #      1100                     - - AAG TTT ACC TAT GCA GGA ATT GAC TGC TCT GC - #G GAA GAA GCA GCC T           3478                                                                       Lys Phe Thr Tyr Ala Gly Ile Asp Cys Ser Al - #a Glu Glu Ala Ala                   1105             - #   1110              - #  1115                         - - AGAGCAGAAC TCTTCATGTA CAACGGCCAT TTCTCCTCAC TGGCGCGAGA GC -             #CTTGACAC   3538                                                                 - - CTGTACCAAG CAAGCCACCC ACTGCCAAGA GATGTGATAT ATAAGTGTAT AT -            #ATTGTGCT   3598                                                                 - - GTGTTTGGGA CCCTCCTCAT ACAGCTCGTG CGGATCTGCA GTGTGTTCTG AC -            #TCTAATGT   3658                                                                 - - GACTGTATAT ACTGCTCGGA GTAAGAATGT GCTAAGATCA GAATGCCTGT TC -            #GTGGTTTC   3718                                                                 - - ATATAATATA TTTTTCTAAA AGCATAGATT GCACAGGAAG GTATGAGTAC AA -            #ATACTGTA   3778                                                                 - - ATGCATAACT TGTTATTGTC CTAGATGTGT TTGACATTTT TCCTTTACAA CT -            #GAATGCTA   3838                                                                 - - TAAAAGTGTT TTGCTGTGTG CGCGTAAGAT ACTGTTCGTT AAAATAAGCA TT -            #CCCTTGAC   3898                                                                 - - AGCACAGGAA GAAAAGCGAG GGAAATGTAT GGATTATATT AAATGTGGGT TA -            #CTACACAA   3958                                                                 - - GAGGCCGAAC ATTCCAAGTA GCAGAAGAGA GGGTCTCTCA ACTCTGCTCC TC -            #ACCTGCAG   4018                                                                 - - AAGCCAGTTT GTTTGGCCAT GTGACAATTG TCCTGTGTTT TTATAGCACC CA -            #AATCATTC   4078                                                                 - - TAAAATATGA ACATCTAAAA ACTTTGCTAG GAGACTAAGA ACCTTTGGAG AG -            #ATAGATAT   4138                                                                 - - AAGTACGGTC AAAAAACAAA ACTGCGCCAT GGTACCC      - #                      - #    4175                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1118 amino - #acids                                               (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Asp Ser Leu Ala Gly Leu Val Leu Cys Gl - #y Val Ser Leu Leu Leu        1               5 - #                 10 - #                 15              - - Tyr Gly Val Val Glu Gly Ala Met Asp Leu Il - #e Leu Ile Asn Ser Leu                   20     - #             25     - #             30                  - - Pro Leu Val Ser Asp Ala Glu Thr Ser Leu Th - #r Cys Ile Ala Ser Gly               35         - #         40         - #         45                      - - Trp His Pro His Glu Pro Ile Thr Ile Gly Ar - #g Asp Phe Glu Ala Leu           50             - #     55             - #     60                          - - Met Asn Gln His Gln Asp Pro Leu Glu Val Th - #r Gln Asp Val Thr Arg       65                 - # 70                 - # 75                 - # 80       - - Glu Trp Ala Lys Lys Val Val Trp Lys Arg Gl - #u Lys Ala Ser Lys Ile                       85 - #                 90 - #                 95              - - Asn Gly Ala Tyr Phe Cys Glu Gly Arg Val Ar - #g Gly Gln Ala Ile Arg                  100      - #           105      - #           110                  - - Ile Arg Thr Met Lys Met Arg Gln Gln Ala Se - #r Phe Leu Pro Ala Thr              115          - #       120          - #       125                      - - Leu Thr Met Thr Val Asp Arg Gly Asp Asn Va - #l Asn Ile Ser Phe Lys          130              - #   135              - #   140                          - - Lys Val Leu Ile Lys Glu Glu Asp Ala Val Il - #e Tyr Lys Asn Gly Ser      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Phe Ile His Ser Val Pro Arg His Glu Val Pr - #o Asp Ile Leu Glu        Val                                                                                             165  - #               170  - #               175             - - His Leu Pro His Ala Gln Pro Gln Asp Ala Gl - #y Val Tyr Ser Ala Arg                  180      - #           185      - #           190                  - - Tyr Ile Gly Gly Asn Leu Phe Thr Ser Ala Ph - #e Thr Arg Leu Ile Val              195          - #       200          - #       205                      - - Arg Arg Cys Glu Ala Gln Lys Trp Gly Pro As - #p Cys Ser Arg Pro Cys          210              - #   215              - #   220                          - - Thr Thr Cys Lys Asn Asn Gly Val Cys His Gl - #u Asp Thr Gly Glu Cys      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ile Cys Pro Pro Gly Phe Met Gly Arg Thr Cy - #s Glu Lys Ala Cys        Glu                                                                                             245  - #               250  - #               255             - - Pro His Thr Phe Gly Arg Thr Cys Lys Glu Ar - #g Cys Ser Gly Pro Glu                  260      - #           265      - #           270                  - - Gly Cys Lys Ser Tyr Val Phe Cys Leu Pro As - #p Pro Tyr Gly Cys Ser              275          - #       280          - #       285                      - - Cys Ala Thr Gly Trp Arg Gly Leu Gln Cys As - #n Glu Ala Cys Pro Ser          290              - #   295              - #   300                          - - Gly Tyr Tyr Gly Pro Asp Cys Lys Leu Arg Cy - #s His Cys Thr Asn Glu      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Glu Ile Cys Asp Arg Phe Gln Gly Cys Leu Cy - #s Ser Gln Gly Trp        Gln                                                                                             325  - #               330  - #               335             - - Gly Leu Gln Cys Glu Lys Glu Gly Arg Pro Ar - #g Met Thr Pro Gln Ile                  340      - #           345      - #           350                  - - Glu Asp Leu Pro Asp His Ile Glu Val Asn Se - #r Gly Lys Phe Asn Pro              355          - #       360          - #       365                      - - Ile Cys Lys Ala Ser Gly Trp Pro Leu Pro Th - #r Ser Glu Glu Met Thr          370              - #   375              - #   380                          - - Leu Val Lys Pro Asp Gly Thr Val Leu Gln Pr - #o Asn Asp Phe Asn Tyr      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Thr Asp Arg Phe Ser Val Ala Ile Phe Thr Va - #l Asn Arg Val Leu        Pro                                                                                             405  - #               410  - #               415             - - Pro Asp Ser Gly Val Trp Val Cys Ser Val As - #n Thr Val Ala Gly Met                  420      - #           425      - #           430                  - - Val Glu Lys Pro Phe Asn Ile Ser Val Lys Va - #l Leu Pro Glu Pro Leu              435          - #       440          - #       445                      - - His Ala Pro Asn Val Ile Asp Thr Gly His As - #n Phe Ala Ile Ile Asn          450              - #   455              - #   460                          - - Ile Ser Ser Glu Pro Tyr Phe Gly Asp Gly Pr - #o Ile Lys Ser Lys Lys      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Leu Phe Tyr Lys Pro Val Asn Gln Ala Trp Ly - #s Tyr Ile Glu Val        Thr                                                                                             485  - #               490  - #               495             - - Asn Glu Ile Phe Thr Leu Asn Tyr Leu Glu Pr - #o Arg Thr Asp Tyr Glu                  500      - #           505      - #           510                  - - Leu Cys Val Gln Leu Ala Arg Pro Gly Glu Gl - #y Gly Glu Gly His Pro              515          - #       520          - #       525                      - - Gly Pro Val Arg Arg Phe Thr Thr Ala Cys Il - #e Gly Leu Pro Pro Pro          530              - #   535              - #   540                          - - Arg Gly Leu Ser Leu Leu Pro Lys Ser Gln Th - #r Ala Leu Asn Leu Thr      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Trp Gln Pro Ile Phe Thr Asn Ser Glu Asp Gl - #u Phe Tyr Val Glu        Val                                                                                             565  - #               570  - #               575             - - Glu Arg Arg Ser Leu Gln Thr Thr Ser Asp Gl - #n Gln Asn Ile Lys Val                  580      - #           585      - #           590                  - - Pro Gly Asn Leu Thr Ser Val Leu Leu Ser As - #n Leu Val Pro Arg Glu              595          - #       600          - #       605                      - - Gln Tyr Thr Val Arg Ala Arg Val Asn Thr Ly - #s Ala Gln Gly Glu Trp          610              - #   615              - #   620                          - - Ser Glu Glu Leu Arg Ala Trp Thr Leu Ser As - #p Ile Leu Pro Pro Gln      625                 6 - #30                 6 - #35                 6 -      #40                                                                              - - Pro Glu Asn Ile Lys Ile Ser Asn Ile Thr As - #p Ser Thr Ala Met        Val                                                                                             645  - #               650  - #               655             - - Ser Trp Thr Ile Val Asp Gly Tyr Ser Ile Se - #r Ser Ile Ile Ile Arg                  660      - #           665      - #           670                  - - Tyr Lys Val Gln Gly Lys Asn Glu Asp Gln Hi - #s Ile Asp Val Lys Ile              675          - #       680          - #       685                      - - Lys Asn Ala Thr Val Thr Gln Tyr Gln Leu Ly - #s Gly Leu Glu Pro Glu          690              - #   695              - #   700                          - - Thr Thr Tyr His Val Asp Ile Phe Ala Glu As - #n Asn Ile Gly Ser Ser      705                 7 - #10                 7 - #15                 7 -      #20                                                                              - - Asn Pro Ala Phe Ser His Glu Leu Arg Thr Le - #u Pro His Ser Pro        Gly                                                                                             725  - #               730  - #               735             - - Ser Ala Asp Leu Gly Gly Gly Lys Met Leu Le - #u Ile Ala Ile Leu Gly                  740      - #           745      - #           750                  - - Ser Ala Gly Met Thr Cys Ile Thr Val Leu Le - #u Ala Phe Leu Ile Met              755          - #       760          - #       765                      - - Leu Gln Leu Lys Arg Ala Asn Val Gln Arg Ar - #g Met Ala Gln Ala Phe          770              - #   775              - #   780                          - - Gln Asn Arg Glu Glu Pro Ala Val Gln Phe As - #n Ser Gly Thr Leu Ala      785                 7 - #90                 7 - #95                 8 -      #00                                                                              - - Leu Asn Arg Lys Ala Lys Asn Asn Pro Asp Pr - #o Thr Ile Tyr Pro        Val                                                                                             805  - #               810  - #               815             - - Leu Asp Trp Asn Asp Ile Lys Ile Gly Glu Gl - #y Asn Phe Gly Gln Val                  820      - #           825      - #           830                  - - Leu Lys Ala Arg Ile Lys Lys Asp Gly Leu Ar - #g Met Asp Ala Ala Ile              835          - #       840          - #       845                      - - Lys Arg Met Lys Glu Tyr Ala Ser Lys Asp As - #p His Arg Asp Phe Ala          850              - #   855              - #   860                          - - Gly Glu Leu Glu Val Leu Cys Lys Leu Gly Hi - #s His Pro Asn Ile Ile      865                 8 - #70                 8 - #75                 8 -      #80                                                                              - - Asn Leu Leu Gly Ala Cys Glu His Arg Gly Ty - #r Leu Tyr Leu Ala        Ile                                                                                             885  - #               890  - #               895             - - Glu Tyr Ala Pro His Gly Asn Leu Leu Asp Ph - #e Leu Arg Lys Ser Arg                  900      - #           905      - #           910                  - - Val Leu Glu Thr Asp Pro Ala Phe Ala Ile Al - #a Asn Ser Thr Ala Ser              915          - #       920          - #       925                      - - Thr Leu Ser Ser Gln Gln Leu Leu His Phe Al - #a Ala Asp Val Ala Arg          930              - #   935              - #   940                          - - Gly Met Asp Tyr Leu Ser Gln Lys Gln Phe Il - #e His Arg Asp Leu Ala      945                 9 - #50                 9 - #55                 9 -      #60                                                                              - - Ala Arg Asn Ile Leu Val Gly Glu Asn Tyr Il - #e Ala Lys Ile Ala        Asp                                                                                             965  - #               970  - #               975             - - Phe Gly Leu Ser Arg Gly Gln Glu Val Tyr Va - #l Lys Lys Thr Met Gly                  980      - #           985      - #           990                  - - Arg Leu Pro Val Arg Trp Met Ala Ile Glu Se - #r Leu Asn Tyr Ser Val              995          - #       1000          - #      1005                     - - Tyr Thr Thr Asn Ser Asp Val Trp Ser Tyr Gl - #y Val Leu Leu Trp Glu          1010             - #   1015              - #  1020                         - - Ile Val Ser Leu Gly Gly Thr Pro Tyr Cys Gl - #y Met Thr Cys Ala Glu      1025                1030 - #                1035 - #               1040        - - Leu Tyr Glu Lys Leu Pro Gln Gly Tyr Arg Le - #u Glu Lys Pro Leu Asn                      1045 - #               1050  - #              1055             - - Cys Asp Asp Glu Val Tyr Asp Leu Met Arg Gl - #n Cys Trp Arg Glu Lys                  1060     - #           1065      - #          1070                 - - Pro Tyr Glu Arg Pro Ser Phe Ala Gln Ile Le - #u Val Ser Leu Asn Arg              1075         - #       1080          - #      1085                     - - Met Leu Glu Glu Arg Lys Thr Tyr Val Asn Th - #r Thr Leu Tyr Glu Lys          1090             - #   1095              - #  1100                         - - Phe Thr Tyr Ala Gly Ile Asp Cys Ser Ala Gl - #u Glu Ala Ala              1105                1110 - #                1115                               - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1601 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Mus muscu - #lus                                                (D) DEVELOPMENTAL STAGE: - #Embryo                                   - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: Murine emb - #ryonic lambda gt10 cDNA library                    (B) CLONE: 1.6kb clone                                               - -   (viii) POSITION IN GENOME:                                                       (A) CHROMOSOME/SEGMENT: 4                                                     (B) MAP POSITION: Betwe - #en the brown and pmv-23 loci              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..903                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - ATC AAG TTT CAA GAC GTG ATC GGA GAG GGC AA - #C TTT GGC CAG GTT CTG           48                                                                       Ile Lys Phe Gln Asp Val Ile Gly Glu Gly As - #n Phe Gly Gln Val Leu             1               5 - #                 10 - #                 15              - - AAG GCA CGC ATC AAG AAG GAT GGG TTA CGG AT - #G GAT GCC GCC ATC AAG           96                                                                       Lys Ala Arg Ile Lys Lys Asp Gly Leu Arg Me - #t Asp Ala Ala Ile Lys                        20     - #             25     - #             30                  - - AGG ATG AAA GAG TAT GCC TCC AAA GAT GAT CA - #C AGG GAC TTC GCA GGA          144                                                                       Arg Met Lys Glu Tyr Ala Ser Lys Asp Asp Hi - #s Arg Asp Phe Ala Gly                    35         - #         40         - #         45                      - - GAA CTG GAG GTT CTT TGT AAA CTT GGA CAC CA - #T CCA AAC ATC ATT AAT          192                                                                       Glu Leu Glu Val Leu Cys Lys Leu Gly His Hi - #s Pro Asn Ile Ile Asn                50             - #     55             - #     60                          - - CTC TTG GGA GCA TGT GAA CAC CGA GGC TAT TT - #G TAC CTA GCT ATT GAG          240                                                                       Leu Leu Gly Ala Cys Glu His Arg Gly Tyr Le - #u Tyr Leu Ala Ile Glu            65                 - # 70                 - # 75                 - # 80       - - TAT GCC CCG CAT GGA AAC CTC CTG GAC TTC CT - #G CGT AAG AGC AGA GTG          288                                                                       Tyr Ala Pro His Gly Asn Leu Leu Asp Phe Le - #u Arg Lys Ser Arg Val                            85 - #                 90 - #                 95              - - CTA GAG ACA GAC CCT GCT TTT GCC ATC GCC AA - #C AGT ACA GCT TCC ACA          336                                                                       Leu Glu Thr Asp Pro Ala Phe Ala Ile Ala As - #n Ser Thr Ala Ser Thr                       100      - #           105      - #           110                  - - CTG TCC TCC CAA CAG CTT CTT CAT TTT GCT GC - #A GAT GTG GCC CGG GGG          384                                                                       Leu Ser Ser Gln Gln Leu Leu His Phe Ala Al - #a Asp Val Ala Arg Gly                   115          - #       120          - #       125                      - - ATG GAC TAC TTG AGC CAG AAA CAG TTT ATC CA - #C AGG GAC CTG GCT GCC          432                                                                       Met Asp Tyr Leu Ser Gln Lys Gln Phe Ile Hi - #s Arg Asp Leu Ala Ala               130              - #   135              - #   140                          - - AGA AAC ATT TTA GTT GGT GAA AAC TAC ATA GC - #C AAA ATA GCA GAT TTT          480                                                                       Arg Asn Ile Leu Val Gly Glu Asn Tyr Ile Al - #a Lys Ile Ala Asp Phe           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - GGA TTG TCA CGA GGT CAA GAA GTG TAT GTG AA - #A AAG ACA ATG GGA        AGG      528                                                                    Gly Leu Ser Arg Gly Gln Glu Val Tyr Val Ly - #s Lys Thr Met Gly Arg                          165  - #               170  - #               175              - - CTC CCA GTG CGT TGG ATG GCA ATC GAA TCA CT - #G AAC TAT AGT GTC TAT          576                                                                       Leu Pro Val Arg Trp Met Ala Ile Glu Ser Le - #u Asn Tyr Ser Val Tyr                       180      - #           185      - #           190                  - - ACA ACC AAC AGT GAT GTC TGG TCC TAT GGT GT - #A TTG CTC TGG GAG ATT          624                                                                       Thr Thr Asn Ser Asp Val Trp Ser Tyr Gly Va - #l Leu Leu Trp Glu Ile                   195          - #       200          - #       205                      - - GTT AGC TTA GGA GGC ACC CCC TAC TGC GGC AT - #G ACG TGC GCG GAG CTC          672                                                                       Val Ser Leu Gly Gly Thr Pro Tyr Cys Gly Me - #t Thr Cys Ala Glu Leu               210              - #   215              - #   220                          - - TAT GAG AAG CTA CCC CAG GGC TAC AGG CTG GA - #G AAG CCC CTG AAC TGT          720                                                                       Tyr Glu Lys Leu Pro Gln Gly Tyr Arg Leu Gl - #u Lys Pro Leu Asn Cys           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - GAT GAT GAG GTG TAT GAT CTA ATG AGA CAG TG - #C TGG AGG GAG AAG        CCT      768                                                                    Asp Asp Glu Val Tyr Asp Leu Met Arg Gln Cy - #s Trp Arg Glu Lys Pro                          245  - #               250  - #               255              - - TAT GAG AGA CCA TCA TTT GCC CAG ATA TTG GT - #G TCC TTA AAC AGG ATG          816                                                                       Tyr Glu Arg Pro Ser Phe Ala Gln Ile Leu Va - #l Ser Leu Asn Arg Met                       260      - #           265      - #           270                  - - CTG GAA GAA CGG AAG ACA TAC GTG AAC ACC AC - #A CTG TAT GAG AAG TTT          864                                                                       Leu Glu Glu Arg Lys Thr Tyr Val Asn Thr Th - #r Leu Tyr Glu Lys Phe                   275          - #       280          - #       285                      - - ACC TAT GCA GGA ATT GAC TGC TCT GCG GAA GA - #A GCA GCC TAGAGCAGAA           913                                                                       Thr Tyr Ala Gly Ile Asp Cys Ser Ala Glu Gl - #u Ala Ala                           290              - #   295              - #   300                          - - CTCTTCATGT ACAACGGCCA TTTCTCCTCA CTGGCGCGAG AGCCTTGACA CC -             #TGTACCAA    973                                                                 - - GCAAGCCACC CACTGCCAAG AGATGTGATA TATAAGTGTA TATATTGTGC TG -            #TGTTTGGG   1033                                                                 - - ACCCTCCTCA TACAGCTCGT GCGGATCTGC AGTGTGTTCT GACTCTAATG TG -            #ACTGTATA   1093                                                                 - - TACTGCTCGG AGTAAGAATG TGCTAAGATC AGAATGCCTG TTCGTGGTTT CA -            #TATAATAT   1153                                                                 - - ATTTTTCTAA AAGCATAGAT TGCACAGGAA GGTATGAGTA CAAATACTGT AA -            #TGCATAAC   1213                                                                 - - TTGTTATTGT CCTAGATGTG TTTGACATTT TTCCTTTACA ACTGAATGCT AT -            #AAAAGTGT   1273                                                                 - - TTTGCTGTGT GCGCGTAAGA TACTGTTCGT TAAAATAAGC ATTCCCTTGA CA -            #GCACAGGA   1333                                                                 - - AGAAAAGCGA GGGAAATGTA TGGATTATAT TAAATGTGGG TTACTACACA AG -            #AGGCCGAA   1393                                                                 - - CATTCCAAGT AGCAGAAGAG AGGGTCTCTC AACTCTGCTC CTCACCTGCA GA -            #AGCCAGTT   1453                                                                 - - TGTTTGGCCA TGTGACAATT GTCCTGTGTT TTTATAGCAC CCAAATCATT CT -            #AAAATATG   1513                                                                 - - AACATCTAAA AACTTTGCTA GGAGACTAAG AACCTTTGGA GAGATAGATA TA -            #AGTACGGT   1573                                                                 - - CAAAAAACAA AACTGCGCCA TGGTACCC         - #                  - #               1601                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 301 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Ile Lys Phe Gln Asp Val Ile Gly Glu Gly As - #n Phe Gly Gln Val Leu        1               5 - #                 10 - #                 15              - - Lys Ala Arg Ile Lys Lys Asp Gly Leu Arg Me - #t Asp Ala Ala Ile Lys                   20     - #             25     - #             30                  - - Arg Met Lys Glu Tyr Ala Ser Lys Asp Asp Hi - #s Arg Asp Phe Ala Gly               35         - #         40         - #         45                      - - Glu Leu Glu Val Leu Cys Lys Leu Gly His Hi - #s Pro Asn Ile Ile Asn           50             - #     55             - #     60                          - - Leu Leu Gly Ala Cys Glu His Arg Gly Tyr Le - #u Tyr Leu Ala Ile Glu       65                 - # 70                 - # 75                 - # 80       - - Tyr Ala Pro His Gly Asn Leu Leu Asp Phe Le - #u Arg Lys Ser Arg Val                       85 - #                 90 - #                 95              - - Leu Glu Thr Asp Pro Ala Phe Ala Ile Ala As - #n Ser Thr Ala Ser Thr                  100      - #           105      - #           110                  - - Leu Ser Ser Gln Gln Leu Leu His Phe Ala Al - #a Asp Val Ala Arg Gly              115          - #       120          - #       125                      - - Met Asp Tyr Leu Ser Gln Lys Gln Phe Ile Hi - #s Arg Asp Leu Ala Ala          130              - #   135              - #   140                          - - Arg Asn Ile Leu Val Gly Glu Asn Tyr Ile Al - #a Lys Ile Ala Asp Phe      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Gly Leu Ser Arg Gly Gln Glu Val Tyr Val Ly - #s Lys Thr Met Gly        Arg                                                                                             165  - #               170  - #               175             - - Leu Pro Val Arg Trp Met Ala Ile Glu Ser Le - #u Asn Tyr Ser Val Tyr                  180      - #           185      - #           190                  - - Thr Thr Asn Ser Asp Val Trp Ser Tyr Gly Va - #l Leu Leu Trp Glu Ile              195          - #       200          - #       205                      - - Val Ser Leu Gly Gly Thr Pro Tyr Cys Gly Me - #t Thr Cys Ala Glu Leu          210              - #   215              - #   220                          - - Tyr Glu Lys Leu Pro Gln Gly Tyr Arg Leu Gl - #u Lys Pro Leu Asn Cys      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Asp Asp Glu Val Tyr Asp Leu Met Arg Gln Cy - #s Trp Arg Glu Lys        Pro                                                                                             245  - #               250  - #               255             - - Tyr Glu Arg Pro Ser Phe Ala Gln Ile Leu Va - #l Ser Leu Asn Arg Met                  260      - #           265      - #           270                  - - Leu Glu Glu Arg Lys Thr Tyr Val Asn Thr Th - #r Leu Tyr Glu Lys Phe              275          - #       280          - #       285                      - - Thr Tyr Ala Gly Ile Asp Cys Ser Ala Glu Gl - #u Ala Ala                      290              - #   295              - #   300                          - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4176 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -      (v) FRAGMENT TYPE: N-terminal                                        - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Mus muscu - #lus                                                (B) STRAIN: CD-1                                                              (D) DEVELOPMENTAL STAGE: - #Embryo                                            (F) TISSUE TYPE: Heart                                               - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: Tek                                                       - -   (viii) POSITION IN GENOME:                                                       (A) CHROMOSOME/SEGMENT: 4                                                     (B) MAP POSITION: Betwe - #en the brown and pmv-23 loci              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 124..3490                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - GCCAACTTGT AAACAAGAGC GAGTGGACCA TGCGAGCGGG AAGTCGCAAA GT -             #TGTGAGTT     60                                                                 - - GTTGAAAGCT TCCCAGGGAC TCATGCTCAT CTGTGGACGC TGGATGGGGA GA -            #TCTGGGGA    120                                                                 - - AGT ATG GAC TCT TTA GCC GGC TTA GTT CTC TG - #T GGA GTC AGC TTG        CTC      168                                                                        Met Asp Ser Leu Ala Gly Leu Val - #Leu Cys Gly Val Ser Leu Leu                 1            - #   5               - #   10               - #   15       - - CTT TAT GGA GTA GTA GAA GGC GCC ATG GAC CT - #G ATC TTG ATC AAT TCC          216                                                                       Leu Tyr Gly Val Val Glu Gly Ala Met Asp Le - #u Ile Leu Ile Asn Ser                            20 - #                 25 - #                 30              - - CTA CCT CTT GTG TCT GAT GCC GAA ACA TCC CT - #C ACC TGC ATT GCC TCT          264                                                                       Leu Pro Leu Val Ser Asp Ala Glu Thr Ser Le - #u Thr Cys Ile Ala Ser                        35     - #             40     - #             45                  - - GGG TGG CAC CCC CAT GAG CCC ATC ACC ATA GG - #A AGG GAC TTT GAA GCC          312                                                                       Gly Trp His Pro His Glu Pro Ile Thr Ile Gl - #y Arg Asp Phe Glu Ala                    50         - #         55         - #         60                      - - TTA ATG AAC CAG CAC CAA GAT CCA CTG GAG GT - #T ACT CAA GAT GTG ACC          360                                                                       Leu Met Asn Gln His Gln Asp Pro Leu Glu Va - #l Thr Gln Asp Val Thr                65             - #     70             - #     75                          - - AGA GAA TGG GCG AAA AAA GTT GTT TGG AAG AG - #A GAA AAG GCC AGT AAG          408                                                                       Arg Glu Trp Ala Lys Lys Val Val Trp Lys Ar - #g Glu Lys Ala Ser Lys            80                 - # 85                 - # 90                 - # 95       - - ATT AAT GGT GCT TAT TTC TGT GAA GGT CGA GT - #T CGA GGA CAG GCT ATA          456                                                                       Ile Asn Gly Ala Tyr Phe Cys Glu Gly Arg Va - #l Arg Gly Gln Ala Ile                           100  - #               105  - #               110              - - AGG ATA CGG ACC ATG AAG ATG CGT CAA CAA GC - #A TCC TTC CTA CCT GCT          504                                                                       Arg Ile Arg Thr Met Lys Met Arg Gln Gln Al - #a Ser Phe Leu Pro Ala                       115      - #           120      - #           125                  - - ACT TTA ACT ATG ACC GTG GAC AGG GGA GAT AA - #T GTG AAC ATA TCT TTC          552                                                                       Thr Leu Thr Met Thr Val Asp Arg Gly Asp As - #n Val Asn Ile Ser Phe                   130          - #       135          - #       140                      - - AAA AAG GTG TTA ATT AAA GAA GAA GAT GCA GT - #G ATT TAC AAA AAT GGC          600                                                                       Lys Lys Val Leu Ile Lys Glu Glu Asp Ala Va - #l Ile Tyr Lys Asn Gly               145              - #   150              - #   155                          - - TCC TTC ATC CAC TCA GTG CCC CGG CAT GAA GT - #A CCT GAT ATT TTA GAA          648                                                                       Ser Phe Ile His Ser Val Pro Arg His Glu Va - #l Pro Asp Ile Leu Glu           160                 1 - #65                 1 - #70                 1 -      #75                                                                              - - GTT CAC TTG CCG CAT GCT CAG CCC CAG GAT GC - #T GGT GTG TAC TCG        GCC      696                                                                    Val His Leu Pro His Ala Gln Pro Gln Asp Al - #a Gly Val Tyr Ser Ala                          180  - #               185  - #               190              - - AGG TAC ATA GGA GGA AAC CTG TTC ACC TCA GC - #C TTC ACC AGG CTG ATT          744                                                                       Arg Tyr Ile Gly Gly Asn Leu Phe Thr Ser Al - #a Phe Thr Arg Leu Ile                       195      - #           200      - #           205                  - - GTT CGG AGA TGT GAA GCT CAG AAG TGG GGG CC - #C GAC TGT AGC CGT CCT          792                                                                       Val Arg Arg Cys Glu Ala Gln Lys Trp Gly Pr - #o Asp Cys Ser Arg Pro                   210          - #       215          - #       220                      - - TGT ACT ACT TGC AAG AAC AAT GGA GTC TGC CA - #T GAA GAT ACC GGG GAA          840                                                                       Cys Thr Thr Cys Lys Asn Asn Gly Val Cys Hi - #s Glu Asp Thr Gly Glu               225              - #   230              - #   235                          - - TGC ATT TGC CCT CCT GGG TTT ATG GGG AGA AC - #A TGT GAG AAA GCT TGT          888                                                                       Cys Ile Cys Pro Pro Gly Phe Met Gly Arg Th - #r Cys Glu Lys Ala Cys           240                 2 - #45                 2 - #50                 2 -      #55                                                                              - - GAG CCG CAC ACA TTT GGC AGG ACC TGT AAA GA - #A AGG TGT AGT GGA        CCA      936                                                                    Glu Pro His Thr Phe Gly Arg Thr Cys Lys Gl - #u Arg Cys Ser Gly Pro                          260  - #               265  - #               270              - - GAA GGA TGC AAG TCT TAT GTG TTC TGT CTC CC - #A GAC CCT TAC GGG TGT          984                                                                       Glu Gly Cys Lys Ser Tyr Val Phe Cys Leu Pr - #o Asp Pro Tyr Gly Cys                       275      - #           280      - #           285                  - - TCC TGT GCC ACA GGC TGG AGG GGG TTG CAG TG - #C AAT GAA GCA TGC CCA         1032                                                                       Ser Cys Ala Thr Gly Trp Arg Gly Leu Gln Cy - #s Asn Glu Ala Cys Pro                   290          - #       295          - #       300                      - - TCT GGT TAC TAC GGA CCA GAC TGT AAG CTC AG - #G TGC CAC TGT ACC AAT         1080                                                                       Ser Gly Tyr Tyr Gly Pro Asp Cys Lys Leu Ar - #g Cys His Cys Thr Asn               305              - #   310              - #   315                          - - GAA GAG ATA TGT GAT CGG TTC CAA GGA TGC CT - #C TGC TCT CAA GGA TGG         1128                                                                       Glu Glu Ile Cys Asp Arg Phe Gln Gly Cys Le - #u Cys Ser Gln Gly Trp           320                 3 - #25                 3 - #30                 3 -      #35                                                                              - - CAA GGG CTG CAG TGT GAG AAA GAA GGC AGG CC - #A AGG ATG ACT CCA        CAG     1176                                                                    Gln Gly Leu Gln Cys Glu Lys Glu Gly Arg Pr - #o Arg Met Thr Pro Gln                          340  - #               345  - #               350              - - ATA GAG GAT TTG CCA GAT CAC ATT GAA GTA AA - #C AGT GGA AAA TTT AAC         1224                                                                       Ile Glu Asp Leu Pro Asp His Ile Glu Val As - #n Ser Gly Lys Phe Asn                       355      - #           360      - #           365                  - - CCC ATC TGC AAA GCC TCT GGG TGG CCA CTA CC - #T ACT AGT GAA GAA ATG         1272                                                                       Pro Ile Cys Lys Ala Ser Gly Trp Pro Leu Pr - #o Thr Ser Glu Glu Met                   370          - #       375          - #       380                      - - ACC CTA GTG AAG CCA GAT GGG ACA GTG CTC CA - #A CCA AAT GAC TTC AAC         1320                                                                       Thr Leu Val Lys Pro Asp Gly Thr Val Leu Gl - #n Pro Asn Asp Phe Asn               385              - #   390              - #   395                          - - TAT ACA GAT CGT TTC TCA GTG GCC ATA TTC AC - #T GTC AAC CGA GTC TTA         1368                                                                       Tyr Thr Asp Arg Phe Ser Val Ala Ile Phe Th - #r Val Asn Arg Val Leu           400                 4 - #05                 4 - #10                 4 -      #15                                                                              - - CCT CCT GAC TCA GGA GTC TGG GTC TGC AGT GT - #G AAC ACA GTG GCT        GGG     1416                                                                    Pro Pro Asp Ser Gly Val Trp Val Cys Ser Va - #l Asn Thr Val Ala Gly                          420  - #               425  - #               430              - - ATG GTG GAA AAG CCT TTC AAC ATT TCC GTC AA - #A GTT CTT CCA GAG CCC         1464                                                                       Met Val Glu Lys Pro Phe Asn Ile Ser Val Ly - #s Val Leu Pro Glu Pro                       435      - #           440      - #           445                  - - CTG CAC GCC CCA AAT GTG ATT GAC ACT GGA CA - #T AAC TTT GCT ATC ATC         1512                                                                       Leu His Ala Pro Asn Val Ile Asp Thr Gly Hi - #s Asn Phe Ala Ile Ile                   450          - #       455          - #       460                      - - AAT ATC AGC TCT GAG CCT TAC TTT GGG GAT GG - #A CCC ATC AAA TCC AAG         1560                                                                       Asn Ile Ser Ser Glu Pro Tyr Phe Gly Asp Gl - #y Pro Ile Lys Ser Lys               465              - #   470              - #   475                          - - AAG CTT TTC TAT AAA CCT GTC AAT CAG GCC TG - #G AAA TAC ATT GAA GTG         1608                                                                       Lys Leu Phe Tyr Lys Pro Val Asn Gln Ala Tr - #p Lys Tyr Ile Glu Val           480                 4 - #85                 4 - #90                 4 -      #95                                                                              - - ACG AAT GAG ATT TTC ACT CTC AAC TAC TTG GA - #G CCG CGG ACT GAC        TAC     1656                                                                    Thr Asn Glu Ile Phe Thr Leu Asn Tyr Leu Gl - #u Pro Arg Thr Asp Tyr                          500  - #               505  - #               510              - - GAG CTG TGT GTG CAG CTG GCC CGT CCT GGA GA - #G GGT GGA GAA GGG CAT         1704                                                                       Glu Leu Cys Val Gln Leu Ala Arg Pro Gly Gl - #u Gly Gly Glu Gly His                       515      - #           520      - #           525                  - - CCT GGG CCT GTG AGA CGA TTT ACA ACA GCG TG - #T ATC GGA CTC CCT CCT         1752                                                                       Pro Gly Pro Val Arg Arg Phe Thr Thr Ala Cy - #s Ile Gly Leu Pro Pro                   530          - #       535          - #       540                      - - CCA AGA GGT CTC AGT CTC CTG CCA AAA AGC CA - #G ACA GCT CTA AAT TTG         1800                                                                       Pro Arg Gly Leu Ser Leu Leu Pro Lys Ser Gl - #n Thr Ala Leu Asn Leu               545              - #   550              - #   555                          - - ACT TGG CAA CCG ATA TTT ACA AAC TCA GAA GA - #T GAA TTT TAT GTG GAA         1848                                                                       Thr Trp Gln Pro Ile Phe Thr Asn Ser Glu As - #p Glu Phe Tyr Val Glu           560                 5 - #65                 5 - #70                 5 -      #75                                                                              - - GTC GAG AGG CGA TCC CTG CAA ACA ACA AGT GA - #T CAG CAG AAC ATC        AAA     1896                                                                    Val Glu Arg Arg Ser Leu Gln Thr Thr Ser As - #p Gln Gln Asn Ile Lys                          580  - #               585  - #               590              - - GTG CCT GGG AAC CTG ACC TCG GTG CTA CTG AG - #C AAC TTA GTC CCC AGG         1944                                                                       Val Pro Gly Asn Leu Thr Ser Val Leu Leu Se - #r Asn Leu Val Pro Arg                       595      - #           600      - #           605                  - - GAG CAG TAC ACA GTC CGA GCT AGA GTC AAC AC - #C AAG GCG CAG GGG GAG         1992                                                                       Glu Gln Tyr Thr Val Arg Ala Arg Val Asn Th - #r Lys Ala Gln Gly Glu                   610          - #       615          - #       620                      - - TGG AGT GAA GAA CTC AGG GCC TGG ACC CTT AG - #T GAC ATT CTC CCT CCT         2040                                                                       Trp Ser Glu Glu Leu Arg Ala Trp Thr Leu Se - #r Asp Ile Leu Pro Pro               625              - #   630              - #   635                          - - CAA CCA GAA AAC ATC AAG ATC TCC AAC ATC AC - #T GAC TCC ACA GCT ATG         2088                                                                       Gln Pro Glu Asn Ile Lys Ile Ser Asn Ile Th - #r Asp Ser Thr Ala Met           640                 6 - #45                 6 - #50                 6 -      #55                                                                              - - GTT TCT TGG ACA ATA GTG GAT GGC TAT TCG AT - #T TCT TCC ATC ATC        ATC     2136                                                                    Val Ser Trp Thr Ile Val Asp Gly Tyr Ser Il - #e Ser Ser Ile Ile Ile                          660  - #               665  - #               670              - - CGG TAT AAG GTT CAG GGC AAA AAT GAA GAC CA - #G CAC ATT GAT GTG AAG         2184                                                                       Arg Tyr Lys Val Gln Gly Lys Asn Glu Asp Gl - #n His Ile Asp Val Lys                       675      - #           680      - #           685                  - - ATC AAG AAT GCT ACC GTT ACT CAG TAC CAG CT - #C AAG GGC CTA GAG CCA         2232                                                                       Ile Lys Asn Ala Thr Val Thr Gln Tyr Gln Le - #u Lys Gly Leu Glu Pro                   690          - #       695          - #       700                      - - GAG ACT ACA TAC CAT GTG GAT ATT TTT GCT GA - #G AAC AAC ATA GGA TCA         2280                                                                       Glu Thr Thr Tyr His Val Asp Ile Phe Ala Gl - #u Asn Asn Ile Gly Ser               705              - #   710              - #   715                          - - AGC AAC CCA GCC TTT TCT CAT GAA CTG AGG AC - #G CTT CCA CAT TCC CCA         2328                                                                       Ser Asn Pro Ala Phe Ser His Glu Leu Arg Th - #r Leu Pro His Ser Pro           720                 7 - #25                 7 - #30                 7 -      #35                                                                              - - GGC TCT GCA GAC CTC GGA GGG GGA AAG ATG CT - #A CTC ATA GCC ATC        CTT     2376                                                                    Gly Ser Ala Asp Leu Gly Gly Gly Lys Met Le - #u Leu Ile Ala Ile Leu                          740  - #               745  - #               750              - - GGG TCG GCT GGA ATG ACT TGC ATC ACC GTG CT - #G TTG GCG TTT CTG ATT         2424                                                                       Gly Ser Ala Gly Met Thr Cys Ile Thr Val Le - #u Leu Ala Phe Leu Ile                       755      - #           760      - #           765                  - - ATG TTG CAA CTG AAG AGA GCA AAT GTC CAA AG - #G AGA ATG GCT CAG GCA         2472                                                                       Met Leu Gln Leu Lys Arg Ala Asn Val Gln Ar - #g Arg Met Ala Gln Ala                   770          - #       775          - #       780                      - - TTC CAG AAC AGA GAA GAA CCA GCT GTG CAG TT - #T AAC TCA GGA ACT CTG         2520                                                                       Phe Gln Asn Arg Glu Glu Pro Ala Val Gln Ph - #e Asn Ser Gly Thr Leu               785              - #   790              - #   795                          - - GCC CTT AAC AGG AAG GCC AAA AAC AAT CCA GA - #T CCC ACA ATT TAT CCT         2568                                                                       Ala Leu Asn Arg Lys Ala Lys Asn Asn Pro As - #p Pro Thr Ile Tyr Pro           800                 8 - #05                 8 - #10                 8 -      #15                                                                              - - GTG CTT GAC TGG AAT GAC ATC AAG TTT CAA GA - #C GTG ATC GGA GAG        GGC     2616                                                                    Val Leu Asp Trp Asn Asp Ile Lys Phe Gln As - #p Val Ile Gly Glu Gly                          820  - #               825  - #               830              - - AAC TTT GGC CAG GTT CTG AAG GCA CGC ATC AA - #G AAG GAT GGG TTA CGG         2664                                                                       Asn Phe Gly Gln Val Leu Lys Ala Arg Ile Ly - #s Lys Asp Gly Leu Arg                       835      - #           840      - #           845                  - - ATG GAT GCC GCC ATC AAG AGG ATG AAA GAG TA - #T GCC TCC AAA GAT GAT         2712                                                                       Met Asp Ala Ala Ile Lys Arg Met Lys Glu Ty - #r Ala Ser Lys Asp Asp                   850          - #       855          - #       860                      - - CAC AGG GAC TTC GCA GGA GAA CTG GAG GTT CT - #T TGT AAA CTT GGA CAC         2760                                                                       His Arg Asp Phe Ala Gly Glu Leu Glu Val Le - #u Cys Lys Leu Gly His               865              - #   870              - #   875                          - - CAT CCA AAC ATC ATT AAT CTC TTG GGA GCA TG - #T GAA CAC CGA GGC TAT         2808                                                                       His Pro Asn Ile Ile Asn Leu Leu Gly Ala Cy - #s Glu His Arg Gly Tyr           880                 8 - #85                 8 - #90                 8 -      #95                                                                              - - TTG TAC CTA GCT ATT GAG TAT GCC CCG CAT GG - #A AAC CTC CTG GAC        TTC     2856                                                                    Leu Tyr Leu Ala Ile Glu Tyr Ala Pro His Gl - #y Asn Leu Leu Asp Phe                          900  - #               905  - #               910              - - CTG CGT AAG AGC AGA GTG CTA GAG ACA GAC CC - #T GCT TTT GCC ATC GCC         2904                                                                       Leu Arg Lys Ser Arg Val Leu Glu Thr Asp Pr - #o Ala Phe Ala Ile Ala                       915      - #           920      - #           925                  - - AAC AGT ACA GCT TCC ACA CTG TCC TCC CAA CA - #G CTT CTT CAT TTT GCT         2952                                                                       Asn Ser Thr Ala Ser Thr Leu Ser Ser Gln Gl - #n Leu Leu His Phe Ala                   930          - #       935          - #       940                      - - GCA GAT GTG GCC CGG GGG ATG GAC TAC TTG AG - #C CAG AAA CAG TTT ATC         3000                                                                       Ala Asp Val Ala Arg Gly Met Asp Tyr Leu Se - #r Gln Lys Gln Phe Ile               945              - #   950              - #   955                          - - CAC AGG GAC CTG GCT GCC AGA AAC ATT TTA GT - #T GGT GAA AAC TAC ATA         3048                                                                       His Arg Asp Leu Ala Ala Arg Asn Ile Leu Va - #l Gly Glu Asn Tyr Ile           960                 9 - #65                 9 - #70                 9 -      #75                                                                              - - GCC AAA ATA GCA GAT TTT GGA TTG TCA CGA GG - #T CAA GAA GTG TAT        GTG     3096                                                                    Ala Lys Ile Ala Asp Phe Gly Leu Ser Arg Gl - #y Gln Glu Val Tyr Val                          980  - #               985  - #               990              - - AAA AAG ACA ATG GGA AGG CTC CCA GTG CGT TG - #G ATG GCA ATC GAA TCA         3144                                                                       Lys Lys Thr Met Gly Arg Leu Pro Val Arg Tr - #p Met Ala Ile Glu Ser                       995      - #           1000      - #          1005                 - - CTG AAC TAT AGT GTC TAT ACA ACC AAC AGT GA - #T GTC TGG TCC TAT GGT         3192                                                                       Leu Asn Tyr Ser Val Tyr Thr Thr Asn Ser As - #p Val Trp Ser Tyr Gly                   1010         - #       1015          - #      1020                     - - GTA TTG CTC TGG GAG ATT GTT AGC TTA GGA GG - #C ACC CCC TAC TGC GGC         3240                                                                       Val Leu Leu Trp Glu Ile Val Ser Leu Gly Gl - #y Thr Pro Tyr Cys Gly               1025             - #   1030              - #  1035                         - - ATG ACG TGC GCG GAG CTC TAT GAG AAG CTA CC - #C CAG GGC TAC AGG CTG         3288                                                                       Met Thr Cys Ala Glu Leu Tyr Glu Lys Leu Pr - #o Gln Gly Tyr Arg Leu           1040                1045 - #                1050 - #               1055        - - GAG AAG CCC CTG AAC TGT GAT GAT GAG GTG TA - #T GAT CTA ATG AGA CAG         3336                                                                       Glu Lys Pro Leu Asn Cys Asp Asp Glu Val Ty - #r Asp Leu Met Arg Gln                           1060 - #               1065  - #              1070             - - TGC TGG AGG GAG AAG CCT TAT GAG AGA CCA TC - #A TTT GCC CAG ATA TTG         3384                                                                       Cys Trp Arg Glu Lys Pro Tyr Glu Arg Pro Se - #r Phe Ala Gln Ile Leu                       1075     - #           1080      - #          1085                 - - GTG TCC TTA AAC AGG ATG CTG GAA GAA CGG AA - #G ACA TAC GTG AAC ACC         3432                                                                       Val Ser Leu Asn Arg Met Leu Glu Glu Arg Ly - #s Thr Tyr Val Asn Thr                   1090         - #       1095          - #      1100                     - - ACA CTG TAT GAG AAG TTT ACC TAT GCA GGA AT - #T GAC TGC TCT GCG GAA         3480                                                                       Thr Leu Tyr Glu Lys Phe Thr Tyr Ala Gly Il - #e Asp Cys Ser Ala Glu               1105             - #   1110              - #  1115                         - - GAA GCA GCC T AGAGCAGAAC TCTTCATGTA CAACGGCCAT TTCT - #CCTCAC               3530                                                                       Glu Ala Ala                                                                   1120                                                                           - - TGGCGCGAGA GCCTTGACAC CTGTACCAAG CAAGCCACCC ACTGCCAAGA GA -             #TGTGATAT   3590                                                                 - - ATAAGTGTAT ATATTGTGCT GTGTTTGGGA CCCTCCTCAT ACAGCTCGTG CG -            #GATCTGCA   3650                                                                 - - GTGTGTTCTG ACTCTAATGT GACTGTATAT ACTGCTCGGA GTAAGAATGT GC -            #TAAGATCA   3710                                                                 - - GAATGCCTGT TCGTGGTTTC ATATAATATA TTTTTCTAAA AGCATAGATT GC -            #ACAGGAAG   3770                                                                 - - GTATGAGTAC AAATACTGTA ATGCATAACT TGTTATTGTC CTAGATGTGT TT -            #GACATTTT   3830                                                                 - - TCCTTTACAA CTGAATGCTA TAAAAGTGTT TTGCTGTGTG CGCGTAAGAT AC -            #TGTTCGTT   3890                                                                 - - AAAATAAGCA TTCCCTTGAC AGCACAGGAA GAAAAGCGAG GGAAATGTAT GG -            #ATTATATT   3950                                                                 - - AAATGTGGGT TACTACACAA GAGGCCGAAC ATTCCAAGTA GCAGAAGAGA GG -            #GTCTCTCA   4010                                                                 - - ACTCTGCTCC TCACCTGCAG AAGCCAGTTT GTTTGGCCAT GTGACAATTG TC -            #CTGTGTTT   4070                                                                 - - TTATAGCACC CAAATCATTC TAAAATATGA ACATCTAAAA ACTTTGCTAG GA -            #GACTAAGA   4130                                                                 - - ACCTTTGGAG AGATAGATAT AAGTACGGTC AAAAAACAAA ACTGCG   - #                   4176                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1122 amino - #acids                                               (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Met Asp Ser Leu Ala Gly Leu Val Leu Cys Gl - #y Val Ser Leu Leu Leu        1               5 - #                 10 - #                 15              - - Tyr Gly Val Val Glu Gly Ala Met Asp Leu Il - #e Leu Ile Asn Ser Leu                   20     - #             25     - #             30                  - - Pro Leu Val Ser Asp Ala Glu Thr Ser Leu Th - #r Cys Ile Ala Ser Gly               35         - #         40         - #         45                      - - Trp His Pro His Glu Pro Ile Thr Ile Gly Ar - #g Asp Phe Glu Ala Leu           50             - #     55             - #     60                          - - Met Asn Gln His Gln Asp Pro Leu Glu Val Th - #r Gln Asp Val Thr Arg       65                 - # 70                 - # 75                 - # 80       - - Glu Trp Ala Lys Lys Val Val Trp Lys Arg Gl - #u Lys Ala Ser Lys Ile                       85 - #                 90 - #                 95              - - Asn Gly Ala Tyr Phe Cys Glu Gly Arg Val Ar - #g Gly Gln Ala Ile Arg                  100      - #           105      - #           110                  - - Ile Arg Thr Met Lys Met Arg Gln Gln Ala Se - #r Phe Leu Pro Ala Thr              115          - #       120          - #       125                      - - Leu Thr Met Thr Val Asp Arg Gly Asp Asn Va - #l Asn Ile Ser Phe Lys          130              - #   135              - #   140                          - - Lys Val Leu Ile Lys Glu Glu Asp Ala Val Il - #e Tyr Lys Asn Gly Ser      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Phe Ile His Ser Val Pro Arg His Glu Val Pr - #o Asp Ile Leu Glu        Val                                                                                             165  - #               170  - #               175             - - His Leu Pro His Ala Gln Pro Gln Asp Ala Gl - #y Val Tyr Ser Ala Arg                  180      - #           185      - #           190                  - - Tyr Ile Gly Gly Asn Leu Phe Thr Ser Ala Ph - #e Thr Arg Leu Ile Val              195          - #       200          - #       205                      - - Arg Arg Cys Glu Ala Gln Lys Trp Gly Pro As - #p Cys Ser Arg Pro Cys          210              - #   215              - #   220                          - - Thr Thr Cys Lys Asn Asn Gly Val Cys His Gl - #u Asp Thr Gly Glu Cys      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ile Cys Pro Pro Gly Phe Met Gly Arg Thr Cy - #s Glu Lys Ala Cys        Glu                                                                                             245  - #               250  - #               255             - - Pro His Thr Phe Gly Arg Thr Cys Lys Glu Ar - #g Cys Ser Gly Pro Glu                  260      - #           265      - #           270                  - - Gly Cys Lys Ser Tyr Val Phe Cys Leu Pro As - #p Pro Tyr Gly Cys Ser              275          - #       280          - #       285                      - - Cys Ala Thr Gly Trp Arg Gly Leu Gln Cys As - #n Glu Ala Cys Pro Ser          290              - #   295              - #   300                          - - Gly Tyr Tyr Gly Pro Asp Cys Lys Leu Arg Cy - #s His Cys Thr Asn Glu      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Glu Ile Cys Asp Arg Phe Gln Gly Cys Leu Cy - #s Ser Gln Gly Trp        Gln                                                                                             325  - #               330  - #               335             - - Gly Leu Gln Cys Glu Lys Glu Gly Arg Pro Ar - #g Met Thr Pro Gln Ile                  340      - #           345      - #           350                  - - Glu Asp Leu Pro Asp His Ile Glu Val Asn Se - #r Gly Lys Phe Asn Pro              355          - #       360          - #       365                      - - Ile Cys Lys Ala Ser Gly Trp Pro Leu Pro Th - #r Ser Glu Glu Met Thr          370              - #   375              - #   380                          - - Leu Val Lys Pro Asp Gly Thr Val Leu Gln Pr - #o Asn Asp Phe Asn Tyr      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Thr Asp Arg Phe Ser Val Ala Ile Phe Thr Va - #l Asn Arg Val Leu        Pro                                                                                             405  - #               410  - #               415             - - Pro Asp Ser Gly Val Trp Val Cys Ser Val As - #n Thr Val Ala Gly Met                  420      - #           425      - #           430                  - - Val Glu Lys Pro Phe Asn Ile Ser Val Lys Va - #l Leu Pro Glu Pro Leu              435          - #       440          - #       445                      - - His Ala Pro Asn Val Ile Asp Thr Gly His As - #n Phe Ala Ile Ile Asn          450              - #   455              - #   460                          - - Ile Ser Ser Glu Pro Tyr Phe Gly Asp Gly Pr - #o Ile Lys Ser Lys Lys      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Leu Phe Tyr Lys Pro Val Asn Gln Ala Trp Ly - #s Tyr Ile Glu Val        Thr                                                                                             485  - #               490  - #               495             - - Asn Glu Ile Phe Thr Leu Asn Tyr Leu Glu Pr - #o Arg Thr Asp Tyr Glu                  500      - #           505      - #           510                  - - Leu Cys Val Gln Leu Ala Arg Pro Gly Glu Gl - #y Gly Glu Gly His Pro              515          - #       520          - #       525                      - - Gly Pro Val Arg Arg Phe Thr Thr Ala Cys Il - #e Gly Leu Pro Pro Pro          530              - #   535              - #   540                          - - Arg Gly Leu Ser Leu Leu Pro Lys Ser Gln Th - #r Ala Leu Asn Leu Thr      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Trp Gln Pro Ile Phe Thr Asn Ser Glu Asp Gl - #u Phe Tyr Val Glu        Val                                                                                             565  - #               570  - #               575             - - Glu Arg Arg Ser Leu Gln Thr Thr Ser Asp Gl - #n Gln Asn Ile Lys Val                  580      - #           585      - #           590                  - - Pro Gly Asn Leu Thr Ser Val Leu Leu Ser As - #n Leu Val Pro Arg Glu              595          - #       600          - #       605                      - - Gln Tyr Thr Val Arg Ala Arg Val Asn Thr Ly - #s Ala Gln Gly Glu Trp          610              - #   615              - #   620                          - - Ser Glu Glu Leu Arg Ala Trp Thr Leu Ser As - #p Ile Leu Pro Pro Gln      625                 6 - #30                 6 - #35                 6 -      #40                                                                              - - Pro Glu Asn Ile Lys Ile Ser Asn Ile Thr As - #p Ser Thr Ala Met        Val                                                                                             645  - #               650  - #               655             - - Ser Trp Thr Ile Val Asp Gly Tyr Ser Ile Se - #r Ser Ile Ile Ile Arg                  660      - #           665      - #           670                  - - Tyr Lys Val Gln Gly Lys Asn Glu Asp Gln Hi - #s Ile Asp Val Lys Ile              675          - #       680          - #       685                      - - Lys Asn Ala Thr Val Thr Gln Tyr Gln Leu Ly - #s Gly Leu Glu Pro Glu          690              - #   695              - #   700                          - - Thr Thr Tyr His Val Asp Ile Phe Ala Glu As - #n Asn Ile Gly Ser Ser      705                 7 - #10                 7 - #15                 7 -      #20                                                                              - - Asn Pro Ala Phe Ser His Glu Leu Arg Thr Le - #u Pro His Ser Pro        Gly                                                                                             725  - #               730  - #               735             - - Ser Ala Asp Leu Gly Gly Gly Lys Met Leu Le - #u Ile Ala Ile Leu Gly                  740      - #           745      - #           750                  - - Ser Ala Gly Met Thr Cys Ile Thr Val Leu Le - #u Ala Phe Leu Ile Met              755          - #       760          - #       765                      - - Leu Gln Leu Lys Arg Ala Asn Val Gln Arg Ar - #g Met Ala Gln Ala Phe          770              - #   775              - #   780                          - - Gln Asn Arg Glu Glu Pro Ala Val Gln Phe As - #n Ser Gly Thr Leu Ala      785                 7 - #90                 7 - #95                 8 -      #00                                                                              - - Leu Asn Arg Lys Ala Lys Asn Asn Pro Asp Pr - #o Thr Ile Tyr Pro        Val                                                                                             805  - #               810  - #               815             - - Leu Asp Trp Asn Asp Ile Lys Phe Gln Asp Va - #l Ile Gly Glu Gly Asn                  820      - #           825      - #           830                  - - Phe Gly Gln Val Leu Lys Ala Arg Ile Lys Ly - #s Asp Gly Leu Arg Met              835          - #       840          - #       845                      - - Asp Ala Ala Ile Lys Arg Met Lys Glu Tyr Al - #a Ser Lys Asp Asp His          850              - #   855              - #   860                          - - Arg Asp Phe Ala Gly Glu Leu Glu Val Leu Cy - #s Lys Leu Gly His His      865                 8 - #70                 8 - #75                 8 -      #80                                                                              - - Pro Asn Ile Ile Asn Leu Leu Gly Ala Cys Gl - #u His Arg Gly Tyr        Leu                                                                                             885  - #               890  - #               895             - - Tyr Leu Ala Ile Glu Tyr Ala Pro His Gly As - #n Leu Leu Asp Phe Leu                  900      - #           905      - #           910                  - - Arg Lys Ser Arg Val Leu Glu Thr Asp Pro Al - #a Phe Ala Ile Ala Asn              915          - #       920          - #       925                      - - Ser Thr Ala Ser Thr Leu Ser Ser Gln Gln Le - #u Leu His Phe Ala Ala          930              - #   935              - #   940                          - - Asp Val Ala Arg Gly Met Asp Tyr Leu Ser Gl - #n Lys Gln Phe Ile His      945                 9 - #50                 9 - #55                 9 -      #60                                                                              - - Arg Asp Leu Ala Ala Arg Asn Ile Leu Val Gl - #y Glu Asn Tyr Ile        Ala                                                                                             965  - #               970  - #               975             - - Lys Ile Ala Asp Phe Gly Leu Ser Arg Gly Gl - #n Glu Val Tyr Val Lys                  980      - #           985      - #           990                  - - Lys Thr Met Gly Arg Leu Pro Val Arg Trp Me - #t Ala Ile Glu Ser Leu              995          - #       1000          - #      1005                     - - Asn Tyr Ser Val Tyr Thr Thr Asn Ser Asp Va - #l Trp Ser Tyr Gly Val          1010             - #   1015              - #  1020                         - - Leu Leu Trp Glu Ile Val Ser Leu Gly Gly Th - #r Pro Tyr Cys Gly Met      1025                1030 - #                1035 - #               1040        - - Thr Cys Ala Glu Leu Tyr Glu Lys Leu Pro Gl - #n Gly Tyr Arg Leu Glu                      1045 - #               1050  - #              1055             - - Lys Pro Leu Asn Cys Asp Asp Glu Val Tyr As - #p Leu Met Arg Gln Cys                  1060     - #           1065      - #          1070                 - - Trp Arg Glu Lys Pro Tyr Glu Arg Pro Ser Ph - #e Ala Gln Ile Leu Val              1075         - #       1080          - #      1085                     - - Ser Leu Asn Arg Met Leu Glu Glu Arg Lys Th - #r Tyr Val Asn Thr Thr          1090             - #   1095              - #  1100                         - - Leu Tyr Glu Lys Phe Thr Tyr Ala Gly Ile As - #p Cys Ser Ala Glu Glu      1105                1110 - #                1115 - #               1120        - - Ala Ala                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Gly Xaa Gly Xaa Xaa Gly                                                  1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Trp Met Ala Ile Glu Ser Leu                                              1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - GACAG TGGGTTCTGG GAGT            - #                  - #                      - # 24                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:10:                       - - CGATGCAGGC AGCTTCTGCG GAT           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - CCTCACCTGC AGAAGCCAGT TTGT          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - GTGGTTTGTC CAACTCATCA ATG           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - CTACCATAAT CCAGTCTACT GC           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 65 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: Jtk14                                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - Ile His Arg Asp Leu Ala Ala Arg Asn Val Le - #u Val Gly Glu Asn Leu      1               5   - #                10  - #                15               - - Ala Ser Lys Ile Ala Asp Phe Gly Leu Ser Ar - #g Gly Glu Glu Val Tyr                  20      - #            25      - #            30                   - - Val Lys Lys Thr Met Gly Arg Leu Pro Val Ar - #g Trp Met Ala Ile Glu              35          - #        40          - #        45                       - - Ser Leu Asn Tyr Ser Val Tyr Thr Thr Lys Se - #r Asp Val Trp Ser Phe          50              - #    55              - #    60                           - - Gly                                                                       - - 65                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 316 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: Ret                                                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - Leu Val Leu Gly Lys Thr Leu Gly Glu Gly Gl - #u Phe Gly Lys Val Val      1               5   - #                10  - #                15               - - Lys Ala Thr Ala Phe His Leu Lys Gly Arg Al - #a Gly Tyr Thr Thr Val                  20      - #            25      - #            30                   - - Ala Val Lys Met Leu Lys Glu Asn Ala Ser Pr - #o Ser Glu Leu Arg Asp              35          - #        40          - #        45                       - - Leu Leu Ser Glu Phe Asn Val Leu Lys Gln Va - #l Asn His Pro His Val          50              - #    55              - #    60                           - - Ile Lys Leu Tyr Gly Ala Cys Ser Gln Asp Gl - #y Pro Leu Leu Leu Ile      65                  - #70                  - #75                  - #80        - - Val Glu Tyr Ala Lys Tyr Gly Ser Leu Arg Gl - #y Phe Leu Arg Glu Ser                      85  - #                90  - #                95               - - Arg Lys Val Gly Pro Gly Tyr Leu Gly Ser Gl - #y Gly Ser Arg Asn Ser                  100      - #           105      - #           110                  - - Ser Ser Leu Asp His Pro Asp Glu Arg Ala Le - #u Thr Met Gly Asp Leu              115          - #       120          - #       125                      - - Ile Ser Phe Ala Trp Gln Ile Ser Gln Gly Me - #t Gln Tyr Leu Ala Glu          130              - #   135              - #   140                          - - Met Lys Leu Val His Arg Asp Leu Ala Ala Ar - #g Asn Ile Leu Val Ala      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Glu Gly Arg Lys Met Lys Ile Ser Asp Phe Gl - #y Leu Ser Arg Asp        Val                                                                                             165  - #               170  - #               175             - - Tyr Glu Glu Asp Pro Tyr Val Lys Arg Ser Gl - #n Gly Arg Ile Pro Val                  180      - #           185      - #           190                  - - Lys Trp Met Ala Ile Glu Ser Leu Phe Asp Hi - #s Ile Tyr Thr Thr Gln              195          - #       200          - #       205                      - - Ser Asp Val Trp Ser Phe Gly Val Leu Leu Tr - #p Glu Ile Val Thr Leu          210              - #   215              - #   220                          - - Gly Gly Asn Pro Tyr Pro Gly Ile Pro Pro Gl - #u Arg Leu Phe Asn Leu      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Leu Lys Thr Gly His Arg Met Glu Arg Pro As - #p Asn Cys Ser Glu        Glu                                                                                             245  - #               250  - #               255             - - Met Tyr Arg Leu Met Leu Gln Cys Trp Lys Gl - #n Glu Pro Asp Lys Arg                  260      - #           265      - #           270                  - - Pro Val Phe Ala Asp Ile Ser Lys Asp Leu Gl - #u Lys Met Met Val Lys              275          - #       280          - #       285                      - - Arg Arg Asp Tyr Leu Asp Leu Ala Ala Ser Th - #r Pro Ser Asp Ser Leu          290              - #   295              - #   300                          - - Ile Tyr Asp Asp Gly Leu Ser Glu Glu Glu Th - #r Pro                      305                 3 - #10                 3 - #15                            - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 313 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: FlgM                                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - Leu Val Leu Gly Lys Pro Leu Gly Glu Gly Cy - #s Phe Gly Gln Val Val      1               5   - #                10  - #                15               - - Leu Ala Glu Ala Ile Gly Leu Asp Lys Asp Ly - #s Pro Asn Arg Val Thr                  20      - #            25      - #            30                   - - Lys Val Ala Val Lys Met Leu Lys Ser Asp Al - #a Thr Glu Lys Asp Leu              35          - #        40          - #        45                       - - Ser Asp Leu Ile Ser Glu Met Glu Met Met Ly - #s Met Ile Gly Lys His          50              - #    55              - #    60                           - - Lys Asn Ile Ile Asn Leu Leu Gly Ala Cys Th - #r Gln Asp Gly Pro Leu      65                  - #70                  - #75                  - #80        - - Tyr Val Ile Val Glu Tyr Ala Ser Lys Gly As - #n Leu Arg Glu Tyr Leu                      85  - #                90  - #                95               - - Gln Ala Arg Arg Pro Pro Gly Leu Glu Tyr Cy - #s Tyr Asn Pro Ser His                  100      - #           105      - #           110                  - - Asn Pro Glu Glu Gln Leu Ser Ser Lys Asp Le - #u Val Ser Cys Ala Tyr              115          - #       120          - #       125                      - - Gln Val Ala Arg Gly Met Glu Tyr Leu Ala Se - #r Lys Lys Cys Ile His          130              - #   135              - #   140                          - - Arg Asp Leu Ala Ala Arg Asn Val Leu Val Th - #r Glu Asp Asn Val Met      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Lys Ile Ala Asp Phe Gly Leu Ala Arg Asp Il - #e His His Ile Asp        Tyr                                                                                             165  - #               170  - #               175             - - Tyr Lys Lys Thr Thr Asn Gly Arg Leu Pro Va - #l Lys Trp Met Ala Pro                  180      - #           185      - #           190                  - - Glu Ala Leu Phe Asp Arg Ile Tyr Thr His Gl - #n Ser Asp Val Trp Ser              195          - #       200          - #       205                      - - Phe Gly Val Leu Leu Trp Glu Ile Phe Thr Le - #u Gly Gly Ser Pro Tyr          210              - #   215              - #   220                          - - Pro Gly Val Pro Val Glu Glu Leu Phe Lys Le - #u Leu Lys Glu Gly His      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Arg Met Asp Lys Pro Ser Asn Cys Thr Asn Gl - #u Leu Tyr Met Met        Met                                                                                             245  - #               250  - #               255             - - Arg Asp Cys Trp His Ala Val Pro Ser Gln Ar - #g Pro Thr Phe Lys Gln                  260      - #           265      - #           270                  - - Leu Val Glu Asp Leu Asp Arg Ile Val Ala Le - #u Thr Ser Asn Gln Glu              275          - #       280          - #       285                      - - Tyr Leu Asp Leu Ser Ile Pro Leu Asp Gln Ty - #r Ser Pro Ser Phe Pro          290              - #   295              - #   300                          - - Asp Thr Arg Ser Ser Thr Cys Ser Ser                                      305                 3 - #10                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: Tek1                                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - Arg Cys Glu Ala Gln Lys Trp Gly Pro Asp Cy - #s Ser Arg Pro Cys Thr      1               5   - #                10  - #                15               - - Thr Cys Lys Asn Asn Gly Val Cys His Glu As - #p Thr Gly Glu Cys Ile                  20      - #            25      - #            30                   - - Cys Pro Pro Gly Phe Met Gly Arg Thr Cys Gl - #u Lys                              35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: Tek2                                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - Ala Cys Glu Pro His Thr Phe Gly Arg Thr Cy - #s Lys Glu Arg Cys Ser      1               5   - #                10  - #                15               - - Gly Pro Glu Gly Cys Lys Ser Tyr Val Phe Cy - #s Leu Pro Asp Pro Tyr                  20      - #            25      - #            30                   - - Gly Cys Ser Cys Ala Thr Gly Trp Arg Gly Le - #u Gln Cys Asn Glu                  35          - #        40          - #        45                       - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: Tek 3                                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - Ala Cys Pro Ser Gly Tyr Tyr Gly Pro Asp Cy - #s Lys Leu Arg Cys His      1               5   - #                10  - #                15               - - Cys Thr Asn Glu Glu Ile Cys Asp Arg Phe Gl - #n Cys Leu Cys Ser Gln                  20      - #            25      - #            30                   - - Gly Trp Gln Gly Leu Gln Cys Glu Lys                                              35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Homo sapi - #ens                                       - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: Tie1                                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - Gly Cys Gly Ala Gly Arg Trp Gly Pro Gly Cy - #s Thr Lys Glu Cys Pro      1               5   - #                10  - #                15               - - Gly Cys Leu His Gly Gly Val Cys His Asp Hi - #s Asp Gly Glu Cys Val                  20      - #            25      - #            30                   - - Cys Pro Pro Gly Phe Thr Gly Thr Arg Cys Gl - #u Gln                              35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Homo sapi - #ens                                       - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: Tie2                                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - Ala Cys Arg Glu Gly Arg Phe Gly Gln Ser Cy - #s Gln Glu Gln Cys Pro      1               5   - #                10  - #                15               - - Gly Ile Ser Gly Cys Arg Gly Leu Thr Phe Cy - #s Leu Pro Asp Pro Tyr                  20      - #            25      - #            30                   - - Gly Cys Ser Cys Gly Ser Gly Trp Arg Gly Se - #r Gln Cys Gln Glu                  35          - #        40          - #        45                       - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Homo sapi - #ens                                       - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: Tie3                                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - Ala Cys Ala Pro Gly His Phe Gly Ala Asp Cy - #s Arg Leu Gln Cys Gln      1               5   - #                10  - #                15               - - Cys Gln Asn Gly Gly Thr Cys Asp Arg Phe Se - #r Gly Cys Val Cys Pro                  20      - #            25      - #            30                   - - Ser Gly Trp His Gly Val His Cys Glu Lys                                          35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: EGF                                                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - - Asn Ser Asp Ser Pro Cys Pro Leu Ser Met As - #p Gly Tyr Cys Leu His      1               5   - #                10  - #                15               - - Asp Gly Val Cys Met Tyr Ile Lys Ala Leu As - #p Lys Tyr Ala Cys Asn                  20      - #            25      - #            30                   - - Cys Val Val Gly Tyr Ile Gly Lys Arg Cys Gl - #n Tyr                              35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: Notch                                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                              - - Gly Arg Tyr Cys Asp Lys Asp Ile Asp Glu Cy - #s Ser Leu Ser Ser Phe      1               5   - #                10  - #                15               - - Cys Arg Asn Gly Ala Ser Cys Leu Asn Val Pr - #o Gly Ser Tyr Arg Cys                  20      - #            25      - #            30                   - - Leu Cys Thr Lys Gly Tyr Glu Gly Arg Asp Cy - #s Ala Ile                          35          - #        40          - #        45                       - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 66 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: TekFn1                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                              - - Glu Pro Tyr Phe Gly Asp Gly Pro Ile Lys Se - #r Lys Lys Leu Phe Tyr      1               5   - #                10  - #                15               - - Lys Pro Val Asn Gln Ala Trp Lys Tyr Ile Gl - #u Val Thr Asn Glu Ile                  20      - #            25      - #            30                   - - Phe Thr Leu Asn Tyr Leu Glu Pro Arg Thr As - #p Tyr Glu Leu Cys Val              35          - #        40          - #        45                       - - Gln Leu Ala Arg Pro Gly Glu Gly Gly Glu Gl - #y His Pro Gly Pro Val          50              - #    55              - #    60                           - - Arg Arg                                                                  65                                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 67 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Homo sapi - #ens                                       - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: TieFn1                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                              - - Phe Ser Gly Asp Gly Pro Ile Ser Thr Val Ar - #g Leu His Tyr Arg Pro      1               5   - #                10  - #                15               - - Gln Asp Ser Thr Met Asp Trp Ser Thr Ile Va - #l Val Asp Pro Ser Glu                  20      - #            25      - #            30                   - - Asn Val Thr Leu Met Asn Leu Arg Pro Lys Th - #r Gly Tyr Ser Val Arg              35          - #        40          - #        45                       - - Val Gln Leu Ser Arg Pro Gly Glu Gly Gly Gl - #u Gly Ala Trp Gly Pro          50              - #    55              - #    60                           - - Pro Thr Leu                                                              65                                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:27:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 99 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: TekFn2                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                              - - Phe Thr Thr Ala Cys Ile Gly Leu Pro Pro Pr - #o Arg Gly Leu Ser Leu      1               5   - #                10  - #                15               - - Leu Pro Lys Ser Gln Thr Ala Leu Asn Leu Th - #r Trp Gln Pro Ile Phe                  20      - #            25      - #            30                   - - Thr Asn Ser Glu Asp Glu Phe Tyr Val Glu Va - #l Glu Arg Arg Ser Leu              35          - #        40          - #        45                       - - Gln Thr Thr Ser Asp Gln Gln Asn Ile Lys Va - #l Pro Gly Asn Leu Thr          50              - #    55              - #    60                           - - Ser Val Leu Leu Ser Asn Leu Val Pro Arg Gl - #u Gln Tyr Thr Val Arg      65                  - #70                  - #75                  - #80        - - Ala Arg Val Asn Thr Lys Ala Gln Gly Glu Tr - #p Ser Glu Glu Leu Arg                      85  - #                90  - #                95               - - Ala Asn Thr                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:28:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 100 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Homo sapi - #ens                                       - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: TieFn2                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                              - - Thr Thr Asp Cys Pro Glu Pro Leu Leu Gln Pr - #o Trp Leu Glu Gly Trp      1               5   - #                10  - #                15               - - His Val Glu Gly Thr Asp Arg Leu Arg Val Se - #r Trp Ser Leu Pro Leu                  20      - #            25      - #            30                   - - Val Pro Gly Pro Leu Val Gly Asp Gly Phe Le - #u Leu Arg Leu Trp Asp              35          - #        40          - #        45                       - - Gly Thr Arg Gly Gln Glu Arg Arg Glu Asn Va - #l Ser Ser Pro Gln Ala          50              - #    55              - #    60                           - - Arg Thr Ala Leu Leu Thr Gly Leu Thr Pro Gl - #y Thr His Tyr Gln Leu      65                  - #70                  - #75                  - #80        - - Asp Val Gln Leu Tyr His Cys Thr Leu Leu Gl - #y Pro Ala Ser Pro Pro                      85  - #                90  - #                95               - - Ala His Val Leu                                                                      100                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:29:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 99 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: TekFn3                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                              - - Leu Ser Asp Ile Leu Pro Pro Gln Pro Glu As - #n Ile Lys Ile Ser Asn      1               5   - #                10  - #                15               - - Ile Thr Asp Ser Thr Ala Met Val Ser Trp Th - #r Ile Val Asp Gly Tyr                  20      - #            25      - #            30                   - - Ser Ile Ser Ser Ile Ile Ile Arg Tyr Lys Va - #l Gln Gly Lys Asn Glu              35          - #        40          - #        45                       - - Asp Gln His Ile Asp Val Lys Ile Lys Asn Al - #a Thr Val Thr Gln Tyr          50              - #    55              - #    60                           - - Gln Leu Lys Gly Leu Glu Pro Glu Thr Thr Ty - #r His Val Asp Ile Phe      65                  - #70                  - #75                  - #80        - - Ala Glu Asn Asn Ile Gly Ser Ser Asn Pro Al - #a Phe Ser Met Glu Leu                      85  - #                90  - #                95               - - Arg Thr Leu                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:30:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 94 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Homo sapi - #ens                                       - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: TieFn3                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                              - - Leu Pro Pro Ser Gly Pro Pro Ala Pro Arg Hi - #s Leu His Ala Gln Ala      1               5   - #                10  - #                15               - - Leu Ser Asp Ser Glu Ile Gln Leu Thr Trp Ly - #s His Pro Glu Ala Leu                  20      - #            25      - #            30                   - - Pro Gly Pro Ile Ser Lys Tyr Val Val Glu Va - #l Gln Val Ala Gly Gly              35          - #        40          - #        45                       - - Ala Gly Asp Pro Leu Trp Ile Asp Val Asp Ar - #g Pro Glu Glu Thr Ser          50              - #    55              - #    60                           - - Thr Ile Ile Arg Gly Leu Asn Ala Ser Thr Ar - #g Tyr Leu Phe Arg Met      65                  - #70                  - #75                  - #80        - - Arg Ala Ser Ile Gln Gly Leu Gly Asp Trp Se - #r Asn Thr Val                              85  - #                90                                      - -  - - (2) INFORMATION FOR SEQ ID NO:31:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 104 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: Finc-rat                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                              - - Val Ser Asp Val Pro Arg Asp Leu Lys Val Il - #e Ala Ser Thr Pro Thr      1               5   - #                10  - #                15               - - Ser Leu Leu Ile Ser Trp Glu Pro Pro Ala Va - #l Ser Val Arg Tyr Tyr                  20      - #            25      - #            30                   - - Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Se - #r Pro Val Gln Lys Phe              35          - #        40          - #        45                       - - Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Il - #e Met Asn Ile Lys Pro          50              - #    55              - #    60                           - - Gly Ala Asp Tyr Thr Ile Thr Leu Tyr Ala Va - #l Thr Gly Arg Gly Asp      65                  - #70                  - #75                  - #80        - - Ser Pro Ala Ser Ser Lys Phe Val Ser Ile As - #n Tyr Gln Thr Lys Ile                      85  - #                90  - #                95               - - Asp Lys Pro Ser Gln Asn Gln Val                                                      100                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:32:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 96 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: DLar                                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                              - - Pro Gly Ala Pro Pro Arg Asn Ile Thr Ala Il - #e Ala Thr Ser Ser Thr      1               5   - #                10  - #                15               - - Thr Ile Ser Leu Ser Trp Leu Pro Pro Pro Va - #l Glu Arg Ser Asn Gly                  20      - #            25      - #            30                   - - Arg Ile Ile Tyr Tyr Lys Val Phe Phe Val Gl - #u Val Gly Arg Glu Asp              35          - #        40          - #        45                       - - Asp Glu Ala Thr Thr Met Thr Leu Asn Met Th - #r Ser Ile Val Leu Asp          50              - #    55              - #    60                           - - Glu Leu Lys Arg Trp Thr Glu Tyr Lys Ile Tr - #p Val Leu Ala Gly Thr      65                  - #70                  - #75                  - #80        - - Ser Val Gly Asp Gly Pro Arg Ser His Pro Il - #e Ile Leu Arg Thr Gln                      85  - #                90  - #                95             __________________________________________________________________________

We claim:
 1. An isolated and purified polypeptide comprising amino acids19 to 744 of SEQ ID NO:
 6. 2. A fusion protein comprising a polypeptideas claimed in claim 1 and a lymphokine or an immunoglobulin molecule. 3.A fusion protein comprising a polypeptide as claimed in claim 1 andinterferon, tumor necrosis factor, Il-1, Il-2, IL-3, Il-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, GM-CSF, CSF-1, or G-CSF.
 4. An isolatedand purified receptor tyrosine kinase protein comprising SEQ ID NO:2,NO:4, or NO:
 6. 5. An isolated and purified protein consisting of theamino acid sequence of amino acids 19 to 744 of SEQ. ID. NO: 6.